Human herpesvirus 6 infection in hematopoietic stem cell transplant patients

Human herpesvirus 6 (HHV-6), a member of the β-herpesvirinae subfamily, shares many biological properties with other members of this family, human herpesvirus 7 and cytomegalovirus. Based on virological analysis, there are two distinct variants (HHV-6A and HHV-6B). While HHV-6B is ubiquitous in the human population, causing exanthem subitum, data regarding HHV-6A remains ambiguous. HHV-6 remains latent in the body after primary infection, and reactivates in immunocompromised patients. HHV-6 infection occurs in nearly half of all stem cell transplant (SCT) recipients within 2–4 weeks following transplantation. Viral infection results in clinical symptoms, including fever, skin rash, pneumonia, bone marrow suppression, encephalitis and rejection. Diagnosis of active HHV-6 infection in SCT recipients can be difficult, as this virus is ubiquitous and persistent in the human body. Therefore, accurate diagnostic measures are required to determine whether the virus is the true pathogen responsible for a clinical event. Although a double-blinded control study will be necessary to elucidate reliable treatment protocols, two anti-viral drugs (ganciclovir and foscarnet) are recommended for the treatment of HHV-6-associated diseases in these patients. Human herpesvirus 6 was discovered as a novel human herpesvirus in the peripheral blood of patients with lymphoproliferative disorders and acquired immunodeficiency syndrome. While it was originally designated ‘human B-lymphotropic virus’ (Salahuddin et al, 1986), subsequent studies established that the virus was primarily T-cell lymphotropic, causing it to be classified as HHV-6 (Lusso et al, 1988). Two genetically distinct variants of the virus exist, HHV-6A and HHV-6B (Schirmer et al, 1991). In most children, primary HHV-6B infection causes exanthem subitum (Yamanishi et al, 1988), a common febrile illness in infants that occurs between 6 months and 1 year of age (Yoshikawa et al, 1989). Several severe complications, including encephalitis/encephalopathy (Asano et al, 1992; Yoshikawa et al, 1992a; Suga et al, 1993), hepatitis (Asano et al, 1990; Tajiri et al, 1997; Ishikawa et al, 2002), and myocarditis (Yoshikawa et al, 2001a), can occur in rare cases following primary HHV-6 infection. This virus probably remains latent in the body after the primary infection, and then reactivates upon host immunosuppression in a manner similar to other human herpesviruses. All previously identified human herpesviruses, especially human cytomegalovirus (HCMV), are important contributors to morbidity and mortality in immunosuppressed organ transplant recipients. As HHV-6 shares many characteristics with HCMV, including DNA sequence homology, elements of genomic organization, antigenic cross-reactivity, and in vitro growth characteristics, HHV-6 may behave similarly as a complication of organ transplantation. Although the pathogenesis of HHV-6 is not well understood, recent evidence suggests that the virus is a serious, potentially life-threatening pathogen in the post-transplant period. This review summarizes the biology of HHV-6 and the clinical features of primary HHV-6 infection. With a focus on HHV-6 infection in haematopoietic SCT, we evaluate the clinical features, route of HHV-6 transmission, diagnostic methods, and important treatments for the management of patients with HHV-6. Human herpesvirus 6 and HHV-7 are recently discovered herpesviruses (Salahuddin et al, 1986; Frenkel et al, 1990), contained within the Roseolovirus genus of the β-herpesvirinae subfamily. HHV-6 and HHV-7 share many properties with ‘HCMV’, another β-herpesvirinae subfamily member, including virion structure, high prevalence in natural host, and the ability to establish latent infection in mononuclear cells and salivary glands. The lytic replication cycle in these viruses is generally longer than in other herpesvirinae subfamilies. Human herpesvirus 6 is an enveloped virion with an icosahedral nucleocapsid of 162 capsomers, enclosing a double-stranded DNA genome. Enveloped extracellular virions range in size from 160 to 200 nm in diameter. Under some fixation conditions, thin section electron microscopy reveals that HHV-6 teguments are smooth; these structures fill the space between the nucleocapsid and envelope, which is characteristic of this virus and is not present in other herpesviruses (Biberfeld et al, 1987). Genomic architecture is similar among the roseoloviruses; genomes contain a central unique segment (U) flanked by a pair of direct repeat structures, DRL and DRR (Lindquester & Pellett, 1991). In comparison with other herpesviruses, roseoloviruses have a low G + C content (35–40%). The HHV-6 genome also contains mammalian telomere-like sequences [(TAACCC)n] at the terminal and junctional DR segments (Gompels & Macaulay, 1995), the function of which is not known. These sequences do not appear to function in either nuclear retention of viral DNA, efficiency of DNA replication, or packaging-associated DNA cleavage (Deng & Dewhurst, 1989). Telomere-like sequences mediate the site-specific integration of the HHV-6 genome, observed proximal to the host chromosome telomere (Luppi et al, 1993; Daibata et al, 1998). Among the roseoloviruses, HHV-6 is closely related to HHV-7, with amino acid sequence identities throughout the genome ranging from 22 to 75% (Nicholas, 1996; Dominguez et al, 1999; Isegawa et al, 1999). HHV-6 has five characteristic genes (DR3, U6, U22, U83 and U94) that distinguish it from HHV-7. HHV-6A and HHV-6B are quite similar, with an overall nucleotide sequence identity of 90% (Dominguez et al, 1999; Isegawa et al, 1999). Variation of the DNA sequences between these strains is less than that between many other herpesvirus strains. Conservation can be even higher at the amino acid level; U94 amino acid sequence is identical among 13 independent HHV-6B isolates (Rapp et al, 2000). The U94 gene is a homologue of the parvovirus rep gene, suggested to play a role in site-specific integration. U94 gene has been suggested as a latency-associated transcript that may block the transcription of other viral genes (Rotola et al, 1998). As the integration of the HHV-6 genome into the human genome has been reported by several institutes (Luppi et al, 1993; Daibata et al, 1998), additional studies will be required to elucidate the function of the U94 gene in viral genome integration. The cellular receptor for HHV-6 entry is CD46 (Santoro et al, 1999), a member of the regulator of complement fixation family. This protein is expressed on the surface of all nucleated human cells. A population of T-cell lines expressing CD46 is unable to support replication of HHV-6A, suggesting the existence of a co-receptor. Several recent reports have demonstrated that binding to both CD46 receptor and the glycoprotein H and L complex are important for entry of the virus into host cells (Mori et al, 2003a,b; Santoro et al, 2003). The HHV-6 replicates within activated CD4 T lymphocytes in vivo (Takahashi et al, 1989; Lusso et al, 1991). HHV-6 can also infect CD8 T lymphocytes and natural killer cells, resulting in the induction of surface CD4 expression and subsequent susceptibility to human immunodeficiency virus (HIV) infection (Lusso et al, 1991, 1993, 1995). CD4 T lymphocytes can be infected concurrently with HHV-6 and HIV. As demonstrated by in vitro infection experiments, HHV-6 can also infect macrophages, dendritic cells, fibroblasts, epithelial cells and bone marrow progenitors (Robert et al, 1996; Kempf et al, 1997; Asada et al, 1999; Luppi et al, 1999; Kakimoto et al, 2002; Yoshikawa et al, 2003a). The results of several in vitro HHV-6 infection experiments have suggested that possible mechanisms for causing HHV-6-associated clinical events include bone marrow suppression (Knox & Carrigan, 1992; Isomura et al, 1997), a skin rash resembling acute graft versus host disease (GVHD) (Yoshikawa et al, 2002a), immunosuppression (Lusso et al, 1993; Gobbi et al, 1999) and encephalitis (He et al, 1996). Latency and reactivation are common biological characteristics of all herpesviruses. After cessation of the primary infection with HHV-6, the viral genome persists within peripheral blood mononuclear cells (PBMCs), particularly adherent cells (monocytes/macrophages) (Kondo et al, 1991). The virus also persists in the salivary glands; viral DNA can frequently be detected in saliva through the use of polymerase chain reaction (PCR) (Cone et al, 1993a). As HHV-6 DNA can also be detected in cerebrospinal fluid (Yoshikawa et al, 1992a; Kondo et al, 1993; Suga et al, 1993) and brain tissue (Luppi et al, 1995; Cuomo et al, 2001; Cermelli et al, 2003), the central nervous system has been postulated as a site of HHV-6 latency. Unfortunately, no reliable animal model is available for the study of HHV-6 reactivation. HHV-6B reactivation from latently infected PBMCs can be induced by infection with HHV-7 in vitro (Katsafanas et al, 1996; Tanaka-Taya et al, 2000). These results support in vivo evidence that demonstrated HHV-6 reactivation at the time of HHV-7 infection (Tanaka-Taya et al, 1994). Latency-associated HHV-6 transcripts encoding open reading flames of the immediate early (IE) proteins IE1 and IE2 have recently been identified (Kondo et al, 2002, 2003). The molecular mechanisms governing HHV-6 latency and reactivation remain poorly understood. Infection with HHV-6B is distributed throughout the world with no seasonal predilection and generally occurs within the first year of life. In contrast to HCMV, most SCT recipients and donors, including children, are generally sero-positive for HHV-6B (Yoshikawa et al, 1991; Wang et al, 1996). The HCMV sero-status of the bone marrow donor and the recipient correlated with the risk of post-transplant HCMV infection. Such factors do not appear to be involved in the frequency of post-transplant HHV-6 infections. The epidemiology of HHV-6A, however, remains unclear. Most infections in either immunocompetent infants or immunocompromised patients are caused by HHV-6B (Drobyski et al, 1993a; Frenkel et al, 1994). HHV-6A has been isolated from immunocompromised patients and patients with neurological complications (Hall et al, 1998). Primary infection with HHV-6B causes exanthem subitum, a common febrile infant disease (Yamanishi et al, 1988). In Japan, 70–80% of infants with primary HHV-6 infection exhibit a typical clinical course of exanthem subitum (Asano et al, 1994). Other groups, however, have reported that only 17% of American children with primary HHV-6 infections develop exanthem subitum; the majority develop undefined febrile illness (Pruksananonda et al, 1992; Hall et al, 1994). The reason underlying this discrepancy remains unclear. Ethnic background may be associated with the typical clinical features in this disease. The clinical course of primary HHV-6B infection is generally benign and self-limiting (Asano et al, 1994). Several severe complications of primary viral infection have been reported, however, including encephalitis/encephalopathy (Asano et al, 1992; Yoshikawa et al, 1992a; Suga et al, 1993), hepatitis (Asano et al, 1990; Tajiri et al, 1997; Ishikawa et al, 2002), thrombocytopenia (Yoshikawa et al, 1993a), hemophagocytic syndrome (Huang et al, 1990), and myocarditis (Yoshikawa et al, 2001a). The clinical features of primary HHV-6A infection remain poorly defined. Most infections in transplant recipients are caused by HHV-6B (Drobyski et al, 1993a; Frenkel et al, 1994), but fatal cases exhibiting primary HHV-6A infection after liver transplantation have been reported (Rossi et al, 2001). There are numerous reports linking additional conditions with HHV-6 infection. As HHV-6 is a ubiquitous virus that persistently infects PBMCs throughout life, it is difficult to determine whether this virus is the primary cause of the clinical feature. The most common procedure for the diagnosis of viral infection is PCR. If clinical samples for PCR examination contain PBMCs harbouring HHV-6 DNA, a false positive PCR test result may occur, detecting only the quiescent viral DNA. Therefore, it is important to choose appropriate samples for PCR examination that do not contain PBMCs, such as serum or cerebrospinal fluid supernatant. The combination of PCR with additional examination methods, including viral isolation and serological assays, are also necessary for the accurate diagnosis of active HHV-6 infection. HHV-6 reactivation occurs in patients with drug-induced hypersensitivity syndrome that exhibit infectious mononucleosis-like symptoms (Suzuki et al, 1998; Tohyama et al, 1998). Correlation between this disease and HHV-6 infection appears to be conclusive, as the virus could be isolated from the patients. Despite the detection of HHV-6 antigen in tissues collected from patients with both multiple sclerosis (Carrigan et al, 1996; Goodman et al, 2003) and pityriasis rosea (Watanabe et al, 2002), further investigation is necessary to demonstrate a link between viral infection and these diseases. The clinical features of HHV-6 infection after SCT suggested by recent studies are summarized in Table I. Biological and molecular analyses indicate that HHV-6 is similar to HCMV. Although the exact frequency of reactivation is difficult to determine, approximately 40–50% of SCT recipients develop HHV-6 infection. The incidence appears to be dependent on the sensitivity of the diagnostic procedures for viral infection. Our recent prospective study on viral isolation analysis determined that about 40% of SCT recipients exhibited HHV-6 viraemia 2–4 weeks after SCT (Yoshikawa et al, 1991, 2002b), which was confirmed by additional studies (Maeda et al, 1999; Imbert-Marcille et al, 2000; Ljungman et al, 2000). Although the frequency of HHV-6 infection after SCT, when diagnosed by PCR, is similar to that after solid organ transplantation, the frequency of HHV-6 viraemia (Yoshikawa et al, 1991, 2002b) is likely to be higher after SCT than after solid organ transplantation (Yoshikawa et al, 1992b, 2000). The risk factors contributing to HHV-6 infection in SCT recipients are not fully understood (Table II). Our recent study demonstrated that the incidence of HHV-6 viraemia is significantly higher among allogeneic SCT recipients than in autologous SCT recipients (P = 0·011). A multivariate analysis of allogeneic SCT recipients revealed that underlying disease (leukaemia or lymphoma) is an independent risk factor (P = 0·02) (Yoshikawa et al, 2002b). Furthermore, the frequency of HHV-6 infection is higher in recipients from unrelated donors than in related donors (Ljungman et al, 2000). In addition, allogeneic bone marrow transplant recipients had a higher frequency of HHV-6 viraemia than allogeneic peripheral blood SCT recipients (Maeda et al, 1999). Sashihara et al (2002) reported that the incidence of HHV-6 infection after cord blood SCT was significantly higher than after either SCT or peripheral blood SCT (P < 0·05). Anti-CD3 monoclonal antibody treatment administered as prophylaxis for acute GVHD also increased the risk of both HHV-6 infection (odds ratio, 2·5; 95% confidence interval, 1·3–4·7) and encephalitis (Zerr et al, 2001). Therefore, SCT recipients with these risk factors should be carefully monitored following the procedure by reliable methods (e.g. real-time PCR). Elucidation of the risk factors for HHV-6 infection is important in the proper management of SCT patients. Several factors, including conditioning regimens, may result in the induction of HHV-6 infection. A multivariate analysis of a large number of cases will be necessary to accurately determine the risk factors for HHV-6 reactivation. Although numerous clinical syndromes have been postulated as HHV-6-associated diseases (Table I), many of these associations have been inconclusive. Therefore, if the physician observes patients with these complications, accurate diagnostic measures, described later in the ‘diagnosis’ section, should be performed to confirm active HHV-6 infection. Four major clinical events, including interstitial pneumonitis, a skin rash resembling acute GVHD, encephalitis and bone marrow suppression, are major prognostic indicators of potential HHV-6 infection and are reviewed in this section. Details of other clinical manifestations including thrombotic microangiopathy (Matsuda et al, 1999) and enteritis (Amo et al, 2003) should be studied from the original papers. Carrigan et al (1991) first reported the association of severe interstitial pneumonitis with HHV-6 infection in two marrow transplant recipients (one autologous SCT and one allogeneic SCT). The virus was repeatedly detected in respiratory specimens from one patient. HHV-6 infected cells were observed in lung tissue from both patients by immunohistochemical staining. Subsequently, Cone et al (1993b) performed a retrospective study of 15 cases of post-SCT pneumonia, 15 accidental deaths and six fetuses. The authors evaluated the quantity of viral DNA in lung tissues collected from each subject by semi-quantifiable PCR analysis, and showed that SCT recipients with idiopathic pneumonia had relatively high of HHV-6 DNA in lung In contrast to two studies study patients allogeneic SCT could not a between HHV-6 infection and pneumonia (Yoshikawa et al, 2002b). In some evidence that HHV-6 infection a role in pneumonia following SCT, the association remains particularly in patients. Human herpesvirus 6 has also been isolated from the blood of SCT recipients 15 after Two of the for acute GVHD, had and rash at the time of virus isolation (Asano et al, 1991). further an association between HHV-6 infection and acute GVHD, we peripheral blood specimens from SCT recipients for the of virus by serological analysis (Yoshikawa et al, 1991). HHV-6 infection was confirmed in of the recipients. Four of the skin with of also a febrile the of viral of the 13 patients these These results that HHV-6 infection may in half of all SCT recipients at approximately weeks following the Viral reactivation may play an important role in acute GVHD or an acute the association between HHV-6 and this skin rash, two institutes have to the virus genome within skin tissues from recipients. These studies that HHV-6 an important role in the of acute GVHD et al, et al, 2003) and the of skin rash after allogeneic SCT et al, 1995; et al, 1998; Cone et al, 1999). In a recent HHV-6 viraemia was in of 15 cases with a skin rash that within 1 of SCT, in comparison with of cases exhibiting a skin rash than 1 after transplantation (P = (Yoshikawa et al, HHV-6 may be involved in the of skin that occur within the first after allogeneic The however, whether HHV-6 causes acute GVHD or the virus causes an illness similar to acute In addition, it is possible that acute GVHD HHV-6 which is not associated with skin HHV-6 infection the of several surface including human antigen and 1 on cells in vitro (Yoshikawa et al, 2003a). The in surface expression may the of cells into causing the skin manifestations in SCT recipients. analysis is to this In contrast to SCT immunocompromised such as infants with acute and liver transplant do not appear to skin primary HHV-6 infection after the (Yoshikawa et al, caused by HHV-6 has been well in immunocompetent children with primary HHV-6 infections. The viral genome has been detected in the cerebrospinal fluid of a of patients (Asano et al, 1992; Yoshikawa et al, 1992a; Suga et al, 1993), suggesting primary In the HHV-6 viral genome could not be detected in cerebrospinal fluid collected from other patients exhibiting neurological symptoms in the suggesting in vitro analysis demonstrated that latent infection and reactivation of HHV-6 the of in cells (Yoshikawa et al, it is that reactivation of the virus in immunocompromised patients may result in the of neurological Several cases of including to HHV-6 infection, have been reported in SCT recipients (Drobyski et al, & 1997; et al, 1998; et al, 1998). allogeneic SCT recipients with monoclonal were likely to develop encephalitis than patients not the treatment (Zerr et al, 2001). As there are no upon examination of patients with central nervous system complications caused by HHV-6, the of patients is There is no in either or cerebrospinal fluid examination in recipients with HHV-6 the of organ transplant recipients with HHV-6 encephalitis also exhibited and within the have been recently et al, 2001; et al, 2001; & 2002; et al, analysis of a fatal HHV-6 encephalitis with detected HHV-6 antigen in the tissues (Drobyski et al, 1994). we recently two SCT recipients with similar clinical features and These features in to the of neurological symptoms weeks after the of and within the may be a diagnostic of HHV-6 as an for encephalitis in SCT recipients. Although the in such cases is generally diagnosis by immediate treatment with or may the of some patients et al, 1998). of viral DNA in cerebrospinal fluid by of PCR is also a for the diagnosis of patients et al, 1999). In contrast to other clinical features, an association between encephalitis/encephalopathy in SCT recipients and HHV-6 infection is likely to be As in infants with primary HHV-6 infection (Yoshikawa et al, 1993a; et al, 2002), idiopathic thrombocytopenia has been in transplant recipients et al, 1995; et al, 1999; Ljungman et al, 2000). In to cell suppression, such as and graft also occurs in such patients (Drobyski et al, Carrigan & et al, 1995; Wang et al, 1996; et al, 1997; et al, 1999; et al, 2003). several have suggested that there is no association between HHV-6 and bone marrow suppression in SCT recipients et al, 1996; Yoshikawa et al, 2002b). Two possible mechanisms have been postulated for bone marrow suppression caused by HHV-6 an by and HHV-6 and direct by the infection of bone marrow progenitors et al, As HHV-6 infection may be associated with severe clinical complications following SCT, it will be important to viral infection accurately and to the route of viral Two likely of HHV-6 infection after SCT reactivation in the recipient and infection acquired from the donor bone Therefore, the latently infected PBMCs of donors and recipients may be an important of viral The of the HHV-6 genome in either the donor or recipient PBMCs to SCT is a of viral infection following the procedure (Yoshikawa et al, 1998). analysis was to HHV-6 strains isolated from a with both and after The analysis suggested that latently infected HHV-6 reactivates in the or of the virus is likely to occur in an immunocompromised (Yoshikawa et al, In as most recipients and donors are for HHV-6, reactivation from recipients and donor marrow may both In a manner similar to HCMV, the HHV-6 viral genome persists in PBMCs throughout after primary infection. of viral genome by of PCR in clinical samples PBMCs may both latent and active viral infection. The available for the detection of HHV-6 infection are summarized in Table Although isolation of the infectious virus remains the procedure for detecting active viral infection, such a is and weeks for As the diagnosis of viral infection is important to establish a treatment the PCR is a As latent HHV-6 infection probably occurs throughout the population, the use of PCR to HHV-6 DNA in blood cells or tissues has in active HHV-6 infection. Two have been to the PCR et al, 1995; et al, 2001; et al, and PCR. with HHV-6 viraemia had higher HHV-6 DNA than HHV-6 viraemia by PCR analysis (Ljungman et al, 2000). PCR a system has been demonstrated to be for active HHV-6 infection in organ transplant recipients et al, 2000; et al, 2000; et al, is difficult, however, to determine that distinguish between active and latent viral infection. of viral by of real-time PCR, which can demonstrate the of viral replication, will important for the management of these patients. As real-time PCR is generally available in many this is the for active viral infection. In to DNA PCR, transcription PCR is another for a diagnostic for detecting active HHV-6 infection et al, 1999; et al, 2001; Yoshikawa et al, clinical studies are to determine whether transcription PCR is a reliable for active viral infection in SCT recipients. As SCT recipients have an the of a serological is to be in these patients. all the SCT recipients with HHV-6 viraemia showed a in HHV-6 antibody in recent analysis serological may also be for the diagnosis of active viral infection in between HHV-6 and HHV-7 have to be et al, 1991; Yoshikawa et al, In addition, an between these viruses has been Although is to determine antibody these the of this to distinguish between HHV-6 and HHV-7 this we demonstrated that a in with an that an major is a serological for the diagnosis of active HHV-6 infection (Yoshikawa et al, Several additional for the detection of have been reported, but a is not detecting HHV-6 in tissues are also Such performed a monoclonal antibody the HHV-6B cells infected with HHV-6. Such an is a reliable for active HCMV infection in SCT recipients. As for HCMV, one has the HHV-6 to active HHV-6 infection in liver transplant recipients et al, 2000). As the number of was to accurately determine the of the methods, a large number of cases should be in In to detecting active HHV-6 infection, another for the accurate diagnosis of HHV-6-associated disease is to determine whether HHV-6 is responsible for the symptoms, as it is necessary to other possible for the and treatment of HHV-6 infection are summarized in Table and are HHV-6 and HHV-7, as in in vitro and other however, are only et al, 1989; & 1990; et al, 1998). The HHV-6 gene the homologue of HCMV & 1999; et al, A HHV-6 exhibiting sensitivity to in vitro was isolated after in the the this was later to in the gene et al, 2001). Although HHV-6 has not been isolated to in of virus is also important for SCT recipients. Although several reports suggesting of and in transplant recipients with severe HHV-6 related diseases have been & 1997; et al, 1998; et al, 2000; et al, 2001; et al, 2001; et al, 2002), no the of treatment HHV-6 infection has been and reviewed cases of HHV-6 encephalitis following bone marrow or solid organ and that of patients were or in comparison with of patients not the et al (2002) also a retrospective analysis to determine whether treatment is for the treatment of HHV-6 encephalitis following SCT recipients with HHV-6-associated central nervous system viral in cerebrospinal fluid with and however, may cause bone marrow suppression and Therefore, the to an Although in vitro studies that HHV-6 is less to Wang et al have suggested the of high prophylaxis for the of HHV-6 infection. As is less than either or the of the as a treatment for HHV-6 infection should be evaluated in a double-blinded control has recently been reported that may HHV-6 in SCT recipients et al, 2002; et al, In contrast to can have severe of the is not recommended for all SCT recipients. If patients at high risk for HHV-6-associated such as encephalitis or bone marrow suppression, could be treatment with may be a for the management of disease in recipients. Furthermore, an appropriate treatment and of should be carefully to establish a reliable treatment with Human herpesvirus 6 can be a pathogen in SCT recipients. Although an association between some post-transplant clinical complications and HHV-6 infection appears to be conclusive, of this association is the this however, of the disease associated with the viral infection is clinical virological in combination with will that can be to the of transplant recipients.