Human herpesvirus 6 and the nervous system

Handbook of Clinical Neurology, Vol. 123 (3rd series) Neurovirology A.C. Tselis and J. Booss, Editors © 2014 Elsevier B.V. All rights reserved

Chapter 16

Human herpesvirus 6 and the nervous system 1

JOSHUA A. HILL1* AND NAGAGOPAL VENNA2 Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA, USA 2

Department of Neurology, Massachusetts General Hospital, Boston, MA, USA

HUMAN HERPESVIRUS 6 AND THE NERVOUS SYSTEM Introduction Human herpesvirus 6 (HHV-6) belongs to the human Herpesviridae family. Herpesviruses share the propensity to infect their host chronically in a latent state with intermittent reactivation, typically occurring during periods of relative immunosuppression. HHV-6 is a member of the b-Herpesviridinae subfamily, including human cytomegalovirus (CMV) and HHV-7. It was first isolated in 1986 from patients with lymphoproliferative disorders (Salahuddin et al., 1986). There are two closely related species, HHV-6A and B, which share approximately 90% homology at the nucleotide level (Braun et al., 1997; Zerr, 2006b). Although HHV-6B is the primary type causing human disease, both species are pathogenic and have differences in tissue distribution and disease association. HHV-6 is a pleiotropic virus capable of infecting a multitude of cell types. It has well-defined neurotropism, with detectable DNA throughout normal brain tissue. Despite its pervasiveness, HHV-6 rarely results in clinical disease and is considered an opportunistic pathogen. Nonetheless, it is implicated in a wide spectrum of diseases (Tables 16.1 and 16.2), including many pathologic processes affecting the central nervous system (CNS). Fatal infections have occurred primarily in severely immunosuppressed patients, although there are reports of deaths due to HHV-6-associated diseases in immuncompetent patients as well. The diversity of associated diseases, if truly caused by HHV-6, may be explained by viral genomic sequence variations affecting cell tropism, host genetics, and patient immune status. The significance of HHV-6 infection of the CNS is often controversial due to the challenge implicit in

attributing disease to a commensal virus of the brain, a theme reiterated throughout this review. As HHV-6 polymerase chain reaction (PCR) assays are positive in 32–85% of brain tissue samples at autopsy (Chan et al., 2001b), the causality of HHV-6 in pathologic derangements of the CNS is difficult to validate. Studies have used a variety of techniques that lack standardization to detect the presence of HHV-6 in an array of specimens, making direct comparisons problematic. In addition, many of the methods employed cannot distinguish between latent and active viral infection, and interpretation of the data requires an understanding of the limitations of each detection method and sample source. However, there is accumulating evidence to suggest that HHV-6 is a real cause of CNS pathology, especially in the setting of immunosuppression.

Biology STRUCTURE Herpesviruses consist of three main structural elements: an icosahedral nucleocapsid that contains the viral DNA genome, an external envelope, and the tegument, consisting of a protein mixture between the nucleocapsid and envelope (Fig. 16.1) (Biberfeld et al., 1987). Mature virions are approximately 200 nm in diameter. The HHV-6 genome is a linear, double-stranded DNA molecule with an overall interstrain nucleotide sequence identity of 90% (Dominguez et al., 1999; Isegawa et al., 1999).

TROPISM HHV-6 is able to infect a wide variety of cell types by binding to the complement regulator receptor CD46, which is ubiquitous in human cell lines (Mori et al., 2003). HHV-6 replicates most efficiently in vitro in

*Correspondence to: Joshua A. Hill, Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA, USA. Tel: þ1-214-500-9243, E-mail: [email protected]

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Table 16.1 Spectrum of clinical diseases caused by human herpesvirus 6 (HHV-6) Immunocompetent

Immunocompromised

Exanthem subitum (roseola, or sixth disease) Hepatic dysfunction and hepatitis Myocarditis Thrombocytopenia Hemophagocytic syndrome CNS dysfunction and disease (see Table 2)

Exanthem subitum-like illness

Pneumonitis Hepatitis Myocarditis Graft dysfunction or rejection after HCT or SOT Associated with acute GvHD, CMV reactivation, fungal infection, increased all-cause mortality after HCT or SOT CNS dysfunction and disease (Table 16.2)

CMV, cytomegalovirus; CNS, central nervous system; GvHD, graft-versus-host disease; HCT, hematopoietic cell transplantation; SOT, solid-organ transplantation.

Table 16.2 Human herpesvirus 6-associated diseases of the central nervous system Febrile seizures Mesial temporal-lobe epilepsy Multiple sclerosis Encephalitis Progressive multifocal leukoencephalopathy Chronic fatigue syndrome Cognitive dysfunction Myelitis

CD4þ T lymphocytes (Takahashi et al., 1989), despite its original name of human B-lymphotropic virus (Salahuddin et al., 1986). In vitro studies have also demonstrated HHV-6 infection of fibroblasts, natural killer cells, continuous liver cells, epithelial cells, endothelial cells, astrocytes, oligodendrocytes, and microglia (De Bolle et al., 2005). This virus is even found in bone marrow progenitor cells, allowing for longitudinal transmission to cells of different lineages such as macrophages and dendritic cells. Within host tissue, HHV-6 has been identified in brain, liver, tonsillar, salivary, and endothelial samples. HHV-6A and B have differences in tropism that may be explained by genomic divergence in the region coding for the viral CD46-binding protein (Mori et al., 2003). Their ability to infect particular T-cell lines, along with other antigenic and biologic properties, determines their

Fig. 16.1. Electron micrograph of human herpesvirus 6 (HHV-6) particles from human progenitor-derived astrocytes infected for 7 days. The three main structural elements include an icosahedral nucleocapsid containing viral DNA, an external envelope, and the tegument between the nucleocapsid and envelope. Mature virions are approximately 200 nm in diameter. (Reproduced with permission from Wainwright et al., 2001.)

classification as distinct species (Flamand et al., 2010). HHV-6A has been shown to replicate more efficiently in various neural cell lines than HHV-6B (De Bolle et al., 2005), and the two species appear to have different tropism within oligodendrocytes (Ahlqvist et al., 2005).

LATENCY Like other HHVs, HHV-6 chronically infects its host after primary infection. It appears that low-level chronic replication occurs in brain tissue and salivary glands (Fox et al., 1990; Chan et al., 2001b; Donati et al., 2003), whereas latency without virion replication occurs in monocytes, peripheral blood mononuclear cells (PBMCs), and bone marrow progenitor cells (Kondo et al., 1991; Luppi et al., 1999). While other herpesviruses remain latent as nuclear episomes, HHV-6 has the unique capacity of integrating into chromosomal telomeres of infected cells (Arbuckle and Medveczky, 2011). Studies have shown that approximately 0.7–2% of infected individuals harbor the complete HHV-6 genome in every cell of their body as a result of integration into germline chromosomes (Ward et al., 2006, 2007; Leong et al., 2007; Pellett et al., 2011). This allows for vertical transmission of chromosomally integrated HHV-6 (ciHHV-6) in a Mendelian manner, with a 50% chance of being passed to a child. All clinical specimens with nucleated cells from these individuals will contain HHV-6 DNA when tested by PCR assays. Chromosomally integrated HHV-6 is suggested by whole blood specimens with persistent HHV-6 DNA detection at levels

HUMAN HERPESVIRUS 6 AND THE NERVOUS SYSTEM > 105.5 copies/mL, which is up to 100-fold higher than levels typically observed during the brief viremia seen in primary infection or reactivation. Even body fluids that are not expected to contain many cells (e.g., serum, plasma, and cerebrospinal fluid (CSF)) may have detectable HHV-6 DNA in patients with ciHHV-6 due to cell lysis during sample preparation.

PATHOGENESIS HHV-6 infection has multiple pathogenic effects. In vitro and in vivo studies have demonstrated apoptosis in lymphocytes and macrophages after acute infection with HHV-6, both from direct and indirect mechanisms (Inoue et al., 1997; Yasukawa et al., 1998; Ichimi et al., 1999; Krueger et al., 2001). HHV-6A may have more cytotoxicity than HHV-6B, as evidenced by increased apoptosis in infected cord blood monocytes (De Bolle et al., 2004b). HHV-6A also appears to have proapoptotic effects in vitro on glial and neuronal cells (Gardell et al., 2006), as well as cytopathic effects in infected oligodendrocytes (Ahlqvist et al., 2005). HHV-6 interacts in a variety of ways with other viruses commonly found in human hosts and with the host immune system. Studies have identified a number of transactivating proteins made by HHV-6 that are capable of regulating steps in the expression of target genes in other pathogens, including human immunodeficiency virus (HIV), Epstein–Barr virus (EBV), adeno-associated virus-2, and human papillomavirus (De Bolle et al., 2005). These proteins may facilitate or inhibit viral replication and infectivity. In addition to this transactivating capacity, HHV-6 has been shown to modulate many aspects of the host’s innate immune response, affecting production and activity of interferons, tumor necrosis factor, interleukins, human leukocyte antigen molecules, and a variety of cell surface receptors. While some of these effects result in T-cell activation, which may be necessary for HHV-6 replication, the T-cell proliferative response to antigen presentation can be severely impaired after viral infection (Horvat et al., 1993; Flamand et al., 1995). This suggests that infection has the capacity to induce immune suppression.

Epidemiology HHV-6 is ubiquitous within the world population; in developed countries, seropositivity for either or both species is >95% (Okuno et al., 1989; Cone et al., 1994). Studies in adults using discriminatory tests for the two species showed a higher prevalence of HHV-6B than A (58% versus 25%) (Wang et al., 1999a, b). Approximately 70–90% of children are infected by 2 years of age, with a peak incidence of acquisition between 9

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and 21 months (Okuno et al., 1989; Zerr et al., 2005b). HHV-6 infection accounts for up to 20% of all cases of acute fever in children of this age group (Hall et al., 1994). Interestingly, primary infection is almost exclusively caused by HHV-6B; the age of seroconversion for HHV-6A is unknown (Dewhurst et al., 1993; Hall et al., 1998). HHV-6 DNA can be detected in divergent regions of the brain in 32–85% of individuals (Luppi et al., 1994, 1995; Chan et al., 2001b; Donati et al., 2003; Boutolleau et al., 2006). HHV-6A and B have been detected postmortem in up to 27.5% and 75% of randomly selected human brain specimens, respectively (Chan et al., 2001b). Given that this ratio is similar to the seroprevalence of each species, the neuroinvasive potential of HHV-6A is similar to HHV-6B. However, a few studies suggest that HHV-6A has greater neurotropism based on increased identification and persistence of this species within the CNS after infection and in the setting of concurrent neurologic symptoms or disease (Hall et al., 1998; Boutolleau et al., 2006). There are a variety of postulated mechanisms of transmission of HHV-6, including intrauterine, perinatal, and fecal–oral spread, none of which seems to be a significant source of infection (Suga et al., 1995b; Maeda et al., 1997; Boutolleau et al., 2003). The most likely vector is via saliva from a child’s mother or other infants, and saliva is a well-documented source of HHV-6 persistence (Mukai et al., 1994; Zerr et al., 2005b). Interestingly, only HHV-6B DNA has been detected in salivary glands (Collot et al., 2002), and the transmission of HHV-6A is not well documented. HHV-6 appears to have a limited range of other susceptible hosts, although antibodies to HHV-6 or a crossreacting virus have been demonstrated in monkeys, and experimental infections in different monkey species have been successful (Higashi et al., 1989; Yalcin et al., 1992). A rodent model for HHV-6 infection does not exist.

Viral detection Diagnosis of clinically relevant HHV-6 infection or reactivation is challenging due to the high prevalence of primary infection with persistence of the virus in a myriad of cell types. Many techniques have been used to identify HHV-6 in human specimens, including serologic studies, antigen detection, isolation by culture, and nucleic acid detection (Table 16.3). Indirect immunofluorescence testing of serum for anti-HHV-6 immunoglobulin G (IgG) and/or IgM is a widely used method to diagnose HHV-6 infection (Zerr, 2006b). This test is labor-intensive, does not discriminate between HHV-6 species, and is limited by

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Table 16.3 Human herpesvirus 6 (HHV-6) diagnostic techniques Assay

Advantages

Serology (antibody detection) Indirect Widely available immunofluorescence Enables serum titer determination

Antibody avidity

Reduced cross-reactivity Discrimination between new and old infection Immunohistochemistry (antigen detection) PBMCs and tissue HHV-6 species discrimination Reflects active infection Culture PBMCs and tissue

HHV-6 species discrimination Reflects active infection PCR* (DNA detection) and RT-PCR* (RNA detection) PCR, plasma HHV-6 species discrimination Reflects active replication Quantitative Rapid result availability PCR, PBMCs HHV-6 species discrimination Rapid result availability

RT-PCR, plasma, and PBMCs LAMP* (DNA detection) Plasma

HHV-6 species discrimination Relfects active replication Quantitative (with plasma) HHV-6 species discrimination Rapid result availability

Disadvantages

Labor-intensive Does not discriminate between HHV-6 species Cross-reactivity with HHV-7 and maternal antibodies Unreliable results in immunocompromised patients Labor-intensive Does not discriminate between HHV-6 species

Labor-intensive Semiquantitative Delayed result availability Labor-intensive Delayed result availability Labor-intensive Expensive

Does not distinguish active from latent infection Unreliable results in lymphopenic patients Expensive Semiquantitative Labor-intensive Expensive

Expensive Semiquantitative

*May be misleading in cases of chromosomally integrated HHV-6. LAMP, loop-mediated isothermal amplification; PBMCs, peripheral blood mononuclear cells; PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR.

cross-reactivity between HHV-6 and -7 antibodies. Interpretation of serologic methods is also complicated by maternal antibodies in the setting of primary infection in infants and the unreliability of antibody titers in severely immunocompromised patients. Improvements in serologic testing now allow for the assessment of the avidity of antibodies for their target antigens whereby recently produced antibodies have lower antigen avidity (indicative of recent infection) and older antibodies have greater avidity (implying infection >6 weeks prior or maternally derived antibodies) (Ward et al., 1993). Avidity assays have a lower likelihood of falsepositive results from cross-reacting antibodies. Active HHV-6 replication can be demonstrated by detection of viral antigens in PBMCs or tissue samples using immunohistochemical techniques (Fig. 16.2) (Tomoiu and Flamand, 2006). This allows for HHV-6

subtype determination with species-specific monoclonal antibodies. Detection of HHV-6 by culture growth is also indicative of active infection and can discriminate between species, but both of these methods involve lengthy procedures that render them unsuitable for routine clinical testing. Viral nucleic acid detection by means of PCR and reverse transcription (RT)-PCR assays have become the method of choice for HHV-6 identification in plasma, serum, and CSF samples (Osiowy et al., 1998; Norton et al., 1999; Van den Bosch et al., 2001; Yoshikawa et al., 2003; Loginov et al., 2009; Lou et al., 2011). Many of these assays are capable of distinguishing between species A and B. PCR techniques have the advantage of speed, specificity, and sensitivity for detection of viral nucleic acid. Primers used for such studies are chosen from a conserved region of the viral

HUMAN HERPESVIRUS 6 AND THE NERVOUS SYSTEM

Fig. 16.2. Active human herpesvirus 6 (HHV-6) infection in the brain is demonstrated by immunohistochemistry of a paraffin-embedded, formalin-fixed, fresh/frozen hippocampal autopsy specimen from a patient with HHV-6-associated encephalitis after hematopoietic cell transplantation. Brown immunoreactivity shows antigen expression detected by immunohistochemistry for the late HHV-6 protein gp116/54/ 64 in neurons (arrows). Magnification 100. (Reproduced from Fotheringham et al., 2007a.)

genome, and the PCR product is identified by gel electrophoresis or hybridization with specific probes (Solbrig et al., 2008). A novel method of loop-mediated isothermal amplification (LAMP) can also rapidly and accurately detect HHV-6A and B DNA, although it is only semiquantitative (Ihira et al., 2004). Detection of viral nucleic acids may indicate active or latent infection depending on the clinical setting and specimen tested. Many studies have shown that detection of HHV-6 DNA in plasma, serum, or CSF correlates well with active viral replication (Suga et al., 1995a; Zerr, 2006a; Fotheringham et al., 2007a). After hematopoietic cell transplantation (HCT), levels of HHV-6 DNA in serum and CSF samples may significantly underestimate the level of viral replication in the brain (Fotheringham et al., 2007a). Quantitative PCR methods have become the most commonly used test, as they allow for fast, sensitive, and absolute quantification of viral load. These tests can be used to establish cut-off points for active infection to improve interpretability, although there are no standardized values indicative of HHV-6 infection or reactivation (Gautheret-Dejean et al., 2002; Ihira et al., 2002; Loginov et al., 2009). Viral DNA identified in white blood cell (WBC) fractions by PCR is difficult to interpret since PBMCs are a site of latency. After HCT, negative results on WBC fractions can also be misleading in lymphopenic patients (Zerr, 2006b). RT-PCR detects messenger (m) RNA and is indicative of active viral replication suggestive of infection, even when PBMCs are tested (Norton et al., 1999). However,

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this method is not readily available in clinical practice. Nested PCR techniques have increased sensitivity and are useful for identifying DNA in cases of very low viral loads (Solbrig et al., 2008). Precaution should be taken when interpreting positive results of HHV-6 tests due to the limitations of each technique and lack of standardization. Furthermore, detection of HHV-6 DNA in any specimen may reflect ciHHV-6 rather than active infection, as discussed above. Excluding primary infection, it was demonstrated that ciHHV-6 (defined by persistent, high-grade HHV-6 DNA detection in plasma or whole blood by quantitative PCR) was the most likely cause of detectable HHV-6 DNA from CSF samples of immunocompetent patients, especially when HHV-6A was identified (Ward et al., 2006, 2007). Limited data indicate that the prevalence of HHV-6 DNA in the CSF due to ciHHV-6 in those with suspected viral CNS infection is the same as that in controls, suggesting that such integration does not predispose to neurologic disease. Although there is evidence that ciHHV-6 can be induced to a state of lytic viral replication, it remains unclear if patients with ciHHV-6 are at higher risk for HHV-6-associated diseases (Pellett et al., 2011). Implicating HHV-6 in disease based on evidence of viral detection in human tissue or fluid samples should not be made without careful consideration of the detection method, clinical context, clinical specimen, and possibility of ciHHV-6. In summary, identification of active HHV-6 replication to suggest clinically significant infection is most accurately demonstrated by culture, immunohistochemical, LAMP, and PCR techniques. Quantitative PCR assays are currently the most useful methods in clinical settings for reasons reviewed above, with appreciation for the possibility of ciHHV-6. All of these techniques are appropriate and more readily available to evaluate for evidence of active HHV-6 infection of in vitro clinical specimens.

SPECTRUM OF CLINICAL DISEASE Immunocompetent host HHV-6 was found to be the causative virus of exanthema subitum (roseola, or sixth disease) in 1988 (Yamanishi et al., 1988; Zerr et al., 2005b), a common illness in children that lasts 3–7 days and typically occurs within the first 2 years of life. Primary infection with HHV-6 results in a benign, self-limited illness in the majority of cases. Most patients develop a mild rash on the trunk, neck, and face (Asano et al., 1994). Associated symptoms may include fever, malaise, inflamed tympanic membranes, and gastrointestinal and respiratory tract symptoms (Hall et al., 1994). FS are reported

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in up to one-third of children with primary HHV-6 infection (Hall et al., 1994; Barone et al., 1995). HHV-6 is a versatile pathogen, and although uncommon, severe complications can occur after acute infection or reactivation in immunocompetent patients. These include liver dysfunction (Tajiri et al., 1990, 1997; Takikawa et al., 1992), fulminant hepatitis (Asano et al., 1990; Mendel et al., 1995; Ishikawa et al., 2002), myocarditis (Yoshikawa et al., 2001; Chang et al., 2009), thrombocytopenia (Saijo et al., 1995; Yoshikawa et al., 1998; Hashimoto et al., 2002), hemophagocytic syndrome (Sugita et al., 1995; Takagi et al., 1996; Portolani et al., 1997), and a variety of CNS diseases. CNS complications will be reviewed in the “CNS disease” section, below. Primary infection with HHV-6 in adults is rare given the high seroprevalence in the population, and it is unclear if the resultant illness has similar manifestations as those seen in children.

Immunocompromised host HHV-6 frequently reactivates in immunosuppressed hosts. Viral DNA is detectable in plasma samples from approximately 50% of patients receiving HCT and 30% of patients undergoing solid-organ transplantation (SOT) within 2–6 weeks of transplantation (Sashihara et al., 2002; Yoshikawa et al., 2002; De Bolle et al., 2005; Wang et al., 2006; Zerr, 2006a; Yamane et al., 2007; Chevallier et al., 2010). HHV-6B is responsible for 97–98% of reactivation events (Drobyski et al., 1993; Wang et al., 1999a, 2006; Boutolleau et al., 2006). As HHV-6 is harbored by many tissues, there is also the possibility of graft-induced (re-)infection with HHV-6 from donor to recipient. Risk factors associated with HHV-6 reactivation are numerous and include unrelated umbilical cord blood transplantation (UCBT), mismatched or unrelated donor, gender-mismatched donor, younger age, timing of HCT other than at the first remission for hematologic malignancy, low pretransplantation anti-HHV-6 IgG titers, and treatment with glucocorticoids (Yoshikawa et al., 2002; Ogata et al., 2006; Zerr, 2006b; Yamane et al., 2007). Complications stemming from HHV-6 infection in immunosuppressed patients are broad. The most frequently reported disease after HCT or SOT is encephalitis (see “CNS disease” section, below), followed by pneumonitis (Cone et al., 1993; Buchbinder et al., 2000; Rapaport et al., 2002; Nishimaki et al., 2003; Bommer et al., 2009; Nakayama et al., 2010), hepatitis (Hill et al., 2014; Ward et al., 1989; Sutherland et al., 1991; Griffiths et al., 1999), and myocarditis (Fukae et al., 2000). Other complications associated with HHV-6 reactivation after HCT or SOT include rash, delayed engraftment, graft rejection, acute graft-versus-host disease

(aGvHD) grade II–IV, CNS disease, and increased allcause mortality (Hoshino et al., 1995; Singh et al., 1997; Lautenschlager et al., 1998; Zerr et al., 2005a, 2011; Betts et al., 2011). CMV reactivation has also been associated with HHV-6 reactivation in SOT patients (Humar et al., 2000; Lehto et al., 2007). Immunomodulatory effects of HHV-6 infection can further predispose already high-risk patients for fungal infections (Dockrell et al., 1999; Rogers et al., 2000b; Boeckh and Nichols, 2003; Jacobs et al., 2003; Lusso, 2006). HHV-6 is also frequently detected in patients with HIV, but its clinical significance remains unclear (Fantry and Cleghorn, 1999). Molecular interactions between these viruses have both positive and negative influences on their replication, but HHV-6 infection does not appear to alter the clinical course of HIV infection (Spira et al., 1990; Nigro et al., 1995; Sever et al., 1995). This virus likely plays a role as an opportunistic agent in HIV/acquired immune deficiency syndrome (AIDS), with active infection resulting in diffuse viral replication in lymph nodes. Cases of pneumonitis (Nigro et al., 1995), retinitis (Fantry and Cleghorn, 1999), and encephalitis (see “CNS disease” section, below) have been reported. Unlike in transplant patients, HHV-6 is a rare cause of morbidity and mortality in the HIV patient population.

Neoplasia The role of HHV-6 in neoplastic events is unclear. Further exploration of its oncogenic potential has been prompted by a developing understanding of the role of other HHVs (EBV, HHV-8) in malignant processes (De Bolle et al., 2005). Establishing a clear connection with HHV-6 and neoplastic or lymphoproliferative disorders has been challenging given the prevalence of this virus in many tissues and its ability to remain in a latent or chromosomally integrated state. Attempts to find an association are further complicated by the altered immune status in patients with malignancies, induced by disease or treatment, which can result in HHV-6 reactivation as an opportunistic rather than a causal pathogen. In light of the discovery of HHV-6 in patients with lymphoproliferative disorders (Salahuddin et al., 1986), there has been investigation for a pathogenic role of HHV-6 in related malignancies. No evidence has been found to support HHV-6 involvement in Hodgkin or non-Hodgkin lymphomas (Rojo et al., 1994; Valente et al., 1996; Luppi et al., 1998). Studies trying to link HHV-6 to cervical cancer were prompted by the discovery of this virus in cervical epithelium (Chen et al., 1994), but no association has been found (Chan et al., 2001a; Tran-Thanh et al., 2002). There is limited evidence

HUMAN HERPESVIRUS 6 AND THE NERVOUS SYSTEM suggesting a pathogenic role of HHV-6 in T-cell chronic lymphoproliferative disease (Braun et al., 1995) and oral carcinoma (Yadav et al., 1997). HHV-6 as a cause of primary CNS malignancies has also been investigated given its neuronal cell tropisms as discussed in the “CNS malignancy” section, below. To date, there is no definitive evidence for HHV-6 as an etiologic agent in neoplasia.

CNS DISEASE Febrile seizures CNS disease due to primary infection by HHV-6 is increasingly recognized. Primary HHV-6 infection coincides with peak incidence of FS in children and has been associated with up to one-third of initial FS (Hall et al., 1994; Barone et al., 1995), as well as recurrent FS and status epilepticus (Kondo et al., 1993; Suga et al., 2000; Ward et al., 2005). Reported detection of HHV-6 in children with FS is variable, ranging from 8% to 40% (Millichap and Millichap, 2006). One study did not find a higher incidence of HHV-6 infection in patients with FS than age-matched controls (Hukin et al., 1998), and it remains unclear if these seizures are due to fever alone versus a direct effect of HHV-6 (or another pathogen) on the brain. HHV-6 DNA is frequently detected in the CSF of immunocompetent infants with acute HHV-6 infection (Kondo et al., 1993; Suga et al., 1993; Caserta et al., 1994). Cases of afebrile convulsions (Zerr et al., 2002b) and meningoencephalitis (Yoshikawa et al., 1992; Suga et al., 1993; Jones et al., 1994; Yoshikawa and Asano, 2000; Kato et al., 2003; Virtanen et al., 2007b) associated with exanthema subitum demonstrate that this virus may have a direct effect on the brain during primary infection. However, HHV-6 DNA was not always detected in the CSF of these patients. Evolving methods of viral detection, as well as reported regional variation in incidence, may affect these results.

Mesial temporal lobe epilepsy Mesial temporal lobe epilepsy (MTLE) is one of the most common and intractable forms of seizure disorders, and HHV-6 has been implicated as a possible cause in both children and adults. Patients with MTLE may be more likely to have a history of FS as infants, a frequent complication of primary HHV-6 infection (Fotheringham et al., 2007b). Hippocampal scarring is a common feature of autopsy specimens in a subset of patients with MTLE and is also a complication of prolonged or complex FS. Imaging studies showing hippocampal atrophy on magnetic resonance imaging (MRI) in MTLE patients have suggested an association with patients who have a

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history of FS (VanLandingham et al., 1998; Theodore et al., 1999). Although the etiology of hippocampal atrophy in MTLE is unclear, HHV-6 demonstrates particular tropism for the hippocampus in histopathologic studies and is associated with hippocampal scarring after encephalitis (Wainwright et al., 2001; De Bolle et al., 2005; Seeley et al., 2007). Up to two-thirds of temporal lobectomy specimens from patients with MTLE showed evidence of active HHV-6B but not HHV-6A replication in hippocampal astrocytes using in situ immunohistochemistry and PCR analysis (Uesugi et al., 2000; Donati et al., 2003; Fotheringham et al., 2007b; Theodore et al., 2008; Li et al., 2011). In comparison, HHV-6 was identified in a paucity of patients with other types of epilepsy. Many of these patients had autopsy findings of hippocampal sclerosis, and higher HHV-6 DNA concentrations were localized to hippocampal astrocytes, along with viral proteins indicative of active replication (Fotheringham et al., 2007b). Further evidence for active HHV-6 infection was demonstrated by growing HHV-6 in cultures of astrocyte cells derived from patients with MTLE. However, unstandardized brain sample selection may confound these findings. Possible pathophysiologic mechanisms for MTLE have been ascribed to glutamate toxicity and transporter dysfunction, as well as astrocyte dysfunction (Danbolt, 2001; Proper et al., 2002; Theodore et al., 2008). Interestingly, superinfection of cultured astrocytes with HHV-6 has correlated with decreased glutamate transporter expression that may result in increased neuronal excitability (Fotheringham et al., 2007b). Perhaps MTLE is related to HHV-6 reactivation within areas of the brain known to harbor latent virus after primary infection in childhood. Alternatively, persistent asymptomatic infection might cause cumulative neurologic injury, leading to the development of epilepsy. These tissue-based and molecular studies provide evidence to support an etiologic link between HHV-6 and MTLE in a subset of patients, although there are likely multiple etiologies for this seizure disorder.

Multiple sclerosis MS is a progressive demyelinating disease characterized by an immune-mediated focal breakdown of myelin sheaths around neuronal axons, causing impaired conduction of nerve impulses and symptoms ranging from blurred vision to paralysis. Pathologic findings include T-cell and macrophage infiltration, astrocytic scar formation, and, eventually, axonal damage and degeneration (Noseworthy et al., 2000). This process is often attributed to an autoimmune phenomenon targeting myelin sheaths produced by oligodendrocytes.

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There are many purported causes of MS, and current theories suggest that a variety of mechanisms may be involved in MS pathology. Infection with viral agents has been implicated as a potential trigger given viral associations with encephalomyelitis and demyelination, as well as the preponderance of CD8 þ T cells (which play an important role in viral immunity) in MS plaques (Babbe et al., 2000). HHVs, in particular, are likely candidates due to their neurotropism and ability to remain latent in infected cells with periodic reactivation. HHV-6B antigen, a marker of active viral protein expression, was first localized to myelin-producing oligodendrocytes in MS lesions in 1995 (Challoner et al., 1995). Since that time, a large archive of conflicting literature exploring the role of HHV-6 in MS has been published. Studies have utilized a myriad of diagnostic techniques in different sample populations and specimens to elucidate the association of HHV-6 with MS, rendering data interpretation challenging.

ANTIGEN AND PCR ASSAYS HHV-6 antigens or DNA localizing to MS plaques was confirmed by others soon after Challoner’s discovery (Friedman et al., 1999; Blumberg et al., 2000b), and HHV-6 DNA was demonstrated to have higher prevalence in these plaques compared to brain tissue from patients with and without MS (Cermelli and Jacobson, 2000; Cermelli et al., 2003). Evidence of HHV-6 replication and mRNA expression localized to MS plaques may also support an etiologic association of this virus with MS (Opsahl and Kennedy, 2005). Furthermore, HHV-6 DNA was found at much higher frequency in MS patients than in controls in an analysis of biopsy specimens from early-manifesting MS lesions, especially in oligodendrocytes and microglia (Goodman et al., 2003). However, HHV-6 antigen was only detected in reactive astrocytes and microglia in this study, but not in myelin-producing oligodendrocytes. The significance of the apparent absence of HHV-6 antigens from infected oligodendrocytes in both acute and chronic MS lesions is uncertain. The authors hypothesize that HHV-6 antigen was only identified in reactive cells that had already phagocytosed HHV-6-infected oligodendrocytes, and they also recognize technical aspects that could have affected these results. Increased rates of HHV-6 DNA or antigen detection in brain specimens are not consistent findings (Mameli et al., 2007). An in situ search of autopsy brain tissues from MS patients for viral transcripts of other herpesivruses, such as EBV, HHV-7, and HHV-8, was also unrevealing (Opsahl and Kennedy, 2006). Antigen detection studies do not provide definitive evidence of HHV-6 as a causal agent in MS, and one must be wary of the

bystander phenomenon, in which immune cells harboring HHV-6 antigens enter the CNS as part of the MS disease process. Limitations of these results also include a lack of systematic selection of brain specimens (which were used on the basis of availability) and sampling sites, difficulty of blinding given characteristic findings of MS plaques, and testing of specimens primarily from patients with progressive MS (Voumvourakis et al., 2010). A number of studies have variably demonstrated an increased burden of HHV-6 DNA in patients with MS using PCR analysis of CSF and peripheral blood samples. HHV-6 DNA detection in CSF was significantly more frequent among MS patients in some studies (Liedtke et al., 1995; Alvarez-Lafuente et al., 2008) and unrevealing in others (Mirandola et al., 1999; Carnero et al., 2002; Gutierrez et al., 2002). Serum PCR studies for HHV-6 DNA similarly showed significantly higher detection in MS patients than controls in some (Akhyani et al., 2000; Alvarez-Lafuente et al., 2002b; Chapenko et al., 2003), but not all, analyses (Mirandola et al., 1999; Alvarez-Lafuente et al., 2006a; Ahram et al., 2009). Among the HHVs, only HHV-6 was found more frequently in PBMCs of MS patients (AlvarezLafuente et al., 2002b). However, these results are difficult to interpret given the inability of this technique to discriminate active from latent infection (Mayne et al., 1998; Rotola et al., 1999; Alvarez-Lafuente et al., 2002a, 2007; Chapenko et al., 2003). The negative study results may argue against a continuous disseminated HHV-6 infection in MS, but they do not rule out a lesion-associated, low-grade infection in the brain. PCR techniques have found an association between MS exacerbations and active HHV-6 infection. Longitudinal studies of MS patients have revealed an increased incidence of HHV-6 DNA detection in plasma or PBMCs during disease relapses, particularly in relapsingremitting MS patients (Berti et al., 2002; Chapenko et al., 2003; Alvarez-Lafuente et al., 2006a, b). Nonetheless, this association also lacks consistency (AlvarezLafuente et al., 2002b).

SEROLOGIC ASSAYS Serum serologic techniques to identify HHV-6 infection also provide conflicting data to implicate HHV-6 as a trigger in the pathogenesis of MS. Elevated levels of IgG (indicative of prior infection) and IgM (indicative of recent infection, reactivation, or ongoing viral replication) antibodies against HHV-6 have been identified in MS patients and are sometimes more prominent early in the disease course (Liedtke et al., 1995; Chapenko et al., 2003; Derfuss et al., 2005; Virtanen et al., 2009; Kitsos et al., 2011). Relapsing-remitting MS in particular

HUMAN HERPESVIRUS 6 AND THE NERVOUS SYSTEM had higher anti-HHV-6 IgM antibodies in some studies, whereas IgG reactivity was stable across all patient groups, as one would expect for a pathogen with high baseline seroprevalence (Soldan et al., 1997; Villoslada et al., 2003). However, a number of reports do not corroborate a higher frequency of serum antibodies against HHV-6 in MS patients (Enbom et al., 1999; Gutierrez et al., 2002; Xu et al., 2002; Kuusisto et al., 2008). Studies evaluating for HHV-6 antibodies in CSF from MS patients found elevated anti-HHV-6 IgG levels compared to controls, suggesting an increased rate of prior CNS infection (Chapenko et al., 2003; Derfuss et al., 2005; Virtanen et al., 2007a; Yao et al., 2008, 2009). Conflicting results have also been obtained that showed no significant difference (Enbom et al., 1999; Gutierrez et al., 2002; Kuusisto et al., 2008). Anti-HHV-6 IgM was rarely isolated in the CSF of MS and control patients in these studies, and no informative comparisons can be made.

VIRUS CULTURE AND LYMPHOPROLIFERATIVE ASSAYS Testing for direct evidence of active infection or immune response using virus isolation or lymphoproliferative techniques has demonstrated a possible etiologic link between MS and HHV-6. HHV-6 viremia identified by virus culture assay was shown to correlate with earlystage MS (Knox and Carrigan, 2000), and a study using lysates from HHV-6A-infected cells revealed a higher frequency of lymphoproliferative responses in MS patients than controls (Soldan et al., 2000).

PATHOGENESIS Immune dysfunction intrinsic to MS may explain the increased prevalence of HHV-6 in these patients, especially during exacerbations. Elevated levels of soluble CD46, the primary cellular receptor for HHV-6, have been described in several autoimmune disorders. A study comparing CD46 and HHV-6 levels in MS patients and controls demonstrated higher levels of CD46 in patients with MS (Soldan et al., 2001). Indeed, this finding correlated with more frequent identification of HHV-6 DNA in serum and CSF samples. HHV-6 may also interact with the immune system to promote the development of MS. Two studies suggest that CCR-2, a chemokine receptor on PBMCs and activated T cells, is an important factor in the development of MS (Izikson et al., 2000; Miyagishi et al., 2003). HHV-6 was found to encode for a highly selective and potent CCR-2 agonist (pU83), and activation of this receptor could result in the recruitment of macrophages and other proinflammatory cells to the site of HHV-6 infection with subsequent formation of MS plaques (Luttichau et al., 2003).

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Support for an HHV-6-mediated autoimmune cause of MS was provided by the discovery of a region of myelin basic protein (a candidate autoantigen) that is identical to residues of the pU24 HHV-6 gene product (TejadaSimon et al., 2003). T-cell and antibody responses to this peptide sequence were elevated in MS patients in this report, invoking molecular mimicry as a potential mechanism for HHV-6-related pathogenesis in the development of MS. However, another study evaluating the ability of HHV-6 to activate T cells capable of crossreacting with myelin basic protein found no significant difference between MS patients and controls (Cirone et al., 2002). Direct neurotoxicity of HHV-6, via induction of soluble factors that enhance necrosis of oligodendrocyte precursor cells and oligodendroglioma cells, has been suggested as a cause of MS lesions (Kong et al., 2003). HHV-6 can also adversely affect repair of CNS demyelination through its effects on oligodendrocytes. In vitro infection of glial precursor cells, prior to maturing into oligodendrocytes, can impair cell replication and other critical cell properties (Dietrich et al., 2004). Some findings suggest that HHV-6A may play a larger role in MS than HHV-6B. Discriminatory studies have shown increased lymphoproliferative responses, serum antibodies, and DNA detection for HHV-6A in MS patients (Soldan et al., 1997, 2000; Akhyani et al., 2000; Kim et al., 2000; Alvarez-Lafuente et al., 2002b). HHV-6A has also been shown to have proapoptotic effects in vitro on glial and neuronal cells, further strengthening a causal association between HHV-6A and MS (Gardell et al., 2006).

ANTIVIRAL TREATMENT The association of HHV-6 with MS may be uncovered by the effect of antiviral medications on the disease. Treatments with antiviral activity have been associated with improvement in MS. Two studies monitoring markers of HHV-6 infection in MS patients receiving interferon-b1a, a common treatment for MS, showed decreased HHV-6 detection in conjunction with decreased MS relapses (Hong et al., 2002; GarciaMontojo et al., 2007). Acyclovir has been shown to reduce the frequency of exacerbations in relapsingremitting MS patients. However, there was no effect on overall neurologic symptom progression, and acyclovir has low efficacy for treatment of HHV-6 (Lycke et al., 1996). Valacyclovir, which has greater efficacy against HHV-6, was also shown to decrease the formation of new lesions in a subset of MS patients with severe disease (Bech et al., 2002). These studies did not evaluate viral response to treatment, however, and other medication effects could confound such associations.

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The cited studies present a diverse array of results acquired utilizing many techniques and sample types in different MS and control populations. Given a lack of test and patient standardization, the conflicting results are very challenging to interpret (Moore and Wolfson, 2002). Detection of antibodies against HHV-6 does not distinguish between active, latent, or chronic persistent infection. Similarly, PCR detection of HHV-6 DNA in PBMCs may have little significance in MS and does not imply causality. Testing in patient groups from geographically diverse populations may further confound study results and comparisons, as MS has genetic predispositions and suspected environmental triggers. Such limitations necessitate more refined studies to understand better the correlation between HHV-6 and MS.

Encephalitis Encephalitis is a well-described complication of HHV-6 infection that primarily occurs in immunosuppressed individuals, although it is also described in the immunocompetent. This is one of the most debilitating and sometimes fatal consequences of HHV-6 infection. HHV-6 causes limbic encephalitis in the medial temporal lobes, with a predilection for the hippocampus (Seeley et al., 2007). The first report of encephalitis due to HHV-6 was published in 1990 and occurred during an episode of exanthema subitum in a 10-month-old boy (Ishiguro et al., 1990).

increasingly recognized. HHV-6-PALE is reported to occur in approximately 1–3% of patients following allogeneic HCT and is generally attributed to viral reactivation of HHV-6B (Fujimaki et al., 2006; Zerr, 2006a; Seeley et al., 2007; Mori et al., 2010; Hill et al., 2012). When stratified by transplant type, the incidence is significantly higher after UCBT with a range from 10–16% versus 1–3% after adult donor PBSC or bone marrow HCT (Ansari et al., 2002; Mori et al., 2010; Hill et al., 2012). HHV-6-PALE has well-defined features that include amnesia (especially anterograde), confusion, the syndrome of inappropriate antidiuretic hormone secretion, mild CSF pleocytosis and protein elevation, seizures, and medial temporal lobe changes on MRI (Table 16.4) (Seeley et al., 2007). In a review of 48 published cases and case series of suspected HHV-6-PALE over a 15-year period, 40 of 48 cases had HHV-6 documented in CSF samples (Zerr, 2006a). All cases followed allogeneic HCT except for 1 after autologous HCT. Symptom onset occurred after engraftment in the majority of cases following adult HCT but often preengraftment after UCBT (Hill et al., 2012). The most common neurologic symptoms were confusion and Table 16.4 Features of human herpesvirus 6 (HHV-6)-associated encephalitis after allogeneic hematopoietic cell transplantation (HCT)

IMMUNOCOMPETENT HOST

Characteristic

Typical findings

HHV-6-associated encephalitis in immunocompetent hosts is less common than in immunocompromised patients. It is primarily described in children presenting with exanthema subitum (Yoshikawa et al., 1992; Suga et al., 1993; Jones et al., 1994; Yoshikawa and Asano, 2000; Kato et al., 2003; Virtanen et al., 2007b), although there are also reports in healthy adults (Birnbaum et al., 2005; Troy et al., 2008). Retrospective PCR and antibody studies of CSF from immunocompetent patients with encephalitis of unclear etiology identified a number of suspected infections due to HHV-6 (McCullers et al., 1995; Patnaik and Peter, 1995; Isaacson et al., 2005). These reports were unable to distinguish between primary infection and viral reactivation. No unifying clinical presentation has been identified among immunocompetent individuals.

Incidence*

1–1.5% of all patients undergoing allogeneic HCT 15–60 days after HCT Confusion, depressed consciousness, anterograde amnesia, seizures Circumscribed, non-enhancing, hyperintense lesions in the medial temporal lobes (especially the hippocampus) HHV-6B DNA (with PCR), mild protein elevation, mild lymphocytic pleocytosis Hyponatremia (due to SIADH) Memory deficits in up to 50% Death due to progressive encephalitis in 2.5–50%

IMMUNOCOMPROMISED HOST

*Incidence after unrelated umbilical cord blood transplantation (UCBT) is 10–15%. { Death due to progressive encephalitis after UCBT is up to 50%. CSF, cerebrospinal fluid; DWI, diffusion-weighted imaging; FLAIR, fluid attenuation inversion recovery; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; SIADH, syndrome of inappropriate antidiuretic hormone secretion.

The first recognized case of HHV-6-associated posttransplantation acute limbic encephalitis (HHV-6-PALE) was reported in 1994 following an allogeneic HCT (Drobyski et al., 1994), and this disease has since been

Disease onset Symptoms

Brain MRI findings (T2, FLAIR, DWI sequences) CSF studies

Serum studies Outcomes{

HUMAN HERPESVIRUS 6 AND THE NERVOUS SYSTEM 337 depressed consciousness beginning at a median of HHV-6-PALE has also been described following 24 days after HCT, with a range of 15–60 days (Zerr, SOT, although with less frequency that is not well 2006a). Clinical seizures were reported in 40% of cases, defined (Bollen et al., 2001; Montejo et al., 2002; although electrographic seizures appeared to occur in a Vinnard et al., 2009). These cases occurred between higher proportion. While computed tomography (CT) of 17 and 90 days following SOT and had similar clinical the brain was often normal, MRI findings were abnorand imaging findings compared with patients undergomal in 30 of 43 cases and involved the medial temporal ing HCT; all patients had positive CSF PCR tests for lobes in 22 of 30 cases. CSF findings were significant for HHV-6. The epidemiology of HHV-6-PALE in this elevated protein levels in 58% of reported cases, as well diverse patient population is unclear at this time. as mild lymphocytic pleocytosis in a minority of patients Encephalitis due to HHV-6 also occurs in HIV(although leukopenia was likely present at the time of infected persons (Knox and Carrigan, 1995; Knox lumbar puncture in many cases). HHV-6B was isolated et al., 1995; Ito et al., 2000; Astriti et al., 2006), although in 24 of 27 cases that underwent discriminatory testing. this disease does not appear to be a frequent complicaRisk factors for HHV-6-PALE are still poorly undertion of HIV/AIDS. HHV-6 DNA was infrequently found stood and are variably reported in the literature. by PCR analysis of CSF samples from HIV patients in Implicated risks include allogeneic HCT, UCBT, younmultiple studies evaluating for viral causes of CNS disger age, mismatched or unrelated donor, genderease, suggesting that it does not play a significant role in mismatched donor, underlying malignancy other than this patient population (Bossolasco et al., 1999; Broccolo hematologic malignancy in first remission or chronic et al., 2000; Quereda et al., 2000). myelogenous leukemia chronic phase, low pretransplant anti-HHV-6 IgG titer, treatment with anti-T-cell CAUSALITY monoclonal antibodies or steroids, high-level plasma HHV-6 viremia, and aGvHD grades II–IV (Ljungman The causal role of HHV-6 in limbic encephalitis is suggested by studies demonstrating HHV-6 DNA in CSF et al., 2000; Zerr et al., 2001; Sashihara et al., 2002; from patients with concurrent evidence of HHV-6 repliYoshikawa et al., 2002; Ogata et al., 2006; Zerr, 2006a; Yamane et al., 2007; Mori et al., 2010). cation in implicated areas of the brain on autopsy specThe majority of cases of HHV-6-PALE have been imens (Drobyski et al., 1994; Wainwright et al., 2001; associated with plasma viremia when tested (Zerr, Fotheringham et al., 2007a; Seeley et al., 2007). Histo2006a; Seeley et al., 2007). Up to 50% of patients receivpathologic findings included HHV-6-infected astrocytes ing adult donor HCT and 80% of patients undergoing and neurons in subcortical areas of diseased white matUCBT reactivate HHV-6 in their plasma (Sashihara ter with neuronal loss, reactive gliosis, demyelination, and lymphocyte infiltration. Active infection with et al., 2002; Yoshikawa et al., 2002; Wang et al., 2006; HHV-6 was further supported in these studies by the Zerr, 2006a; Yamane et al., 2007; Chevallier et al., 2010). Recipients of UCBT also develop plasma finding of HHV-6 mRNA and proteins produced only HHV-6 viral loads approximately 1 log10 higher than during viral replication within the hippocampus and the adult donor cell recipients, likely due to a variety of other limbic structures, but not elsewhere in the brain. factors specific to UCBT that result in increased immune The hippocampus had the highest burden of disease in dysfunction (Hill et al., 2012). Higher HHV-6 plasma most samples, and reasons for its particular susceptibilviral loads are associated with an increased risk of ity to HHV-6 are unknown. Despite the high blood reacHHV-6-PALE, with levels typically 100-fold greater tivation rate of HHV-6 after HCT, one study showed among patients developing this disease compared to that CSF PCR testing was positive in only 1 of 107 immuother viremic patients (Ogata et al., 2006). A threshold nocompromised patients without CNS symptoms but plasma viral load of 104 copies/mL has been identified positive in 5 of 11 patients who experienced CNS sympbelow which HHV-6-PALE did not occur in a case series toms of unknown etiology (Wang et al., 1999c). Other employing weekly PCR testing (Ogata et al., 2006, 2010). studies have demonstrated that active HHV-6 replication These studies, along with another (Hill et al., 2012), found within brain parenchyma is underestimated by CSF viral peak plasma HHV-6 viral loads  105 copies/mL to be loads, purporting an underappreciation of viral reactivaapproximately 71% sensitive and 94% specific for a diagtion in clinical settings (Fotheringham et al., 2007a). nosis of HHV-6-PALE; specificity increased to 98% for As with other disease associations, detection of viral loads  106 copies/mL. In the latter study, results HHV-6 in clinical samples must be interpreted with caushould be interpreted with caution, as plasma HHV-6 tion given the possibility of ciHHV-6, in which the findviral load testing was not routinely checked in all patients ing of HHV-6 DNA does not necessarily reflect active but rather clinically driven when the treating clinician disease (see “Biology: Latency” and “Viral detection” had concern for HHV-6 reactivation or reinfection. sections above).

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Fig. 16.3. Magnetic resonance (MR) images from 2 patients with human herpesvirus 6 (HHV-6)-associated posttransplantation acute limbic encephalitis. (A) Axial fluid attenuation inversion recovery (FLAIR) MR image showing characteristic well-demarcated, high-intensity signal abnormalities in the bilateral medial temporal lobes involving the hippocampus and amygdala. (B) Coronal FLAIR MR image of the hippocampi heads with L > R hyperintensity.

IMAGING Imaging abnormalities associated with HHV-6 encephalitis have well-defined features. Brain CT scans in cases of exanthema subitum in children with CNS complications have demonstrated changes, including cerebral edema and hypodensities in the cortex, thalami, cerebellum, and brainstem (Yoshikawa et al., 1992; Sloots et al., 1993; Oki et al., 1995; Yanagihara et al., 1995). Subsequent cases utilizing MRI in immunocompetent and immunocompromised children revealed similar signal abnormalities in diverse areas throughout the brain, with a preponderance of involvement in the medial temporal lobes and especially the thalamic and hippocampal structures (Kimura and Nezu, 1998; Nagasawa et al., 2007; Yoshinari et al., 2007; Provenzale et al., 2008, 2010; Crawford et al., 2009a). Imaging abnormalities in immunocompromised adults with HHV-6-PALE also have similar findings. Although brain CT is typically normal, MRI often reveals well-circumscribed, hyperintense, nonenhancing lesions involving the medial temporal lobes, and especially the hippocampus, on T2, fluid attenuation inversion recovery (FLAIR) and diffusion-weighted

MRI sequences (Fig. 16.3) (Wainwright et al., 2001; Fujimaki et al., 2006; Gorniak et al., 2006; Noguchi et al., 2006; Zerr, 2006a; Seeley et al., 2007; Gewurz et al., 2008; Vinnard et al., 2009). These findings are often bilateral but may be unilateral. A diverse set of extrahippocampal structures in the limbic neuroanatomic distribution can also be affected, including the amygdala, entorhinal cortex, hypothalamus, and deep forebrain structures (Gewurz et al., 2008; Provenzale et al., 2008, 2010). Extrahippocampal changes are seen more frequently among children and after UCBT, for unclear reasons. Imaging studies in patients with HHV-6-associated encephalitis may be normal in up to 30% of cases, especially when performed early in the disease course (Zerr, 2006a). Repeat scanning, if obtained, may show interval development of disease. Follow-up imaging in both children and adults has sometimes shown chronic necrotization or atrophy of affected regions several months after initial studies (Gorniak et al., 2006; Crawford et al., 2007, 2009a; Seeley et al., 2007). While the radiographic features of HHV-6-associated encephalitis are not specific and have significant overlap with herpes simplex virus, among other infectious agents,

HUMAN HERPESVIRUS 6 AND THE NERVOUS SYSTEM medial temporal lobe changes with involvement of the hippocampus and other limbic structures are highly suspicious for HHV-6 infection in the appropriate clinical setting.

OUTCOMES Neurologic recovery after HHV-6-associated encephalitis is variable. In cases among children with primary infection, outcomes range from complete recovery to persistent neurologic sequelae in up to 47% of patients, including spastic quadriplegia and mental retardation, as well as death in 2.5% (Yoshikawa et al., 2009). A retrospective analysis of 23 cases of HHV-6-PALE showed that clinical status appeared to improve after antiviral treatment in 91% of patients, but 43% of patients had persistent mild-to-moderate memory deficits (Muta et al., 2009). Another large review found that 43% of 44 patients with informative outcomes appeared to make a full recovery, 18% improved but had residual neurologic symptoms, 14% showed improvement but died from other conditions, and 25% appeared to have progressive encephalitis that may have resulted in death (Zerr, 2006a). Although a minority of patients with HHV-6-PALE following adult donor HCT have died due to progressive CNS disease, cases after UCBT have up to 50% mortality (Hill et al., 2012). Given the severity of HHV-6-associated encephalitis, centers experienced in caring for patients with this disease advocate a pre-emptive approach to treating suspected cases after primary infection (Hennus et al., 2009) and following HCT or SOT (Gewurz et al., 2008). Although a small prospective study did not demonstrate a short-term survival benefit to routine surveillance for blood HHV-6 reactivation early after HCT (Betts et al., 2011), it is reasonable to send intermittent plasma surveillance assays for HHV-6 viral load in such patients (Gewurz et al., 2008). CSF testing for HHV-6 DNA should be performed in all high-risk patients who develop neurologic symptoms that are not obviously attributable to another cause. Since these results are often unavailable for a few days, one should consider initiating empiric antiviral therapy for suspected cases in the appropriate clinical context. There are no prospective clinical trials evaluating treatment strategies in patients with HHV-6-PALE, and available antiviral agents with activity against HHV-6 have high side-effect profiles. Accordingly, a full course of treatment should ideally be guided by clear laboratory evidence of HHV-6 infection with CSF PCR testing. These patients also require close monitoring for seizure activity and prophylactic use of antiseizure drugs should be considered. Treatment is further discussed in the “Treatment” section, below.

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Progressive multifocal leukoencephalopathy PML is a progressive demyelinating disease of the CNS caused by JC polyoma virus (JCV) reactivation. This disease occurs almost exclusively in the setting of impaired cell-mediated immunity, most commonly due to HIV/ AIDS. PCR and immunohistochemical techniques have identified HHV-6 DNA and evidence of viral replication in some demyelinative lesions of PML, with co-localization of HHV-6 and JCV DNA and antigens to the same oligodendrocytes (Mock et al., 1999; Blumberg et al., 2000a; Daibata et al., 2001). In fact, a greater frequency of HHV-6 than JCV genomes was detected in oligodendrocytes within PML lesions, without involvement of adjacent, healthy tissue (Mock et al., 1999). Lesser amounts of HHV-6 DNA were detected in control brain tissue affected by a variety of other diseases, and no HHV-6 antigens were identified in control tissues that included individuals with HIV encephalopathy but without PML. However, CSF PCR analysis for HHV-6 DNA in a study of HIVinfected patients with neurologic disorders, 36 of whom had histopathologic evidence of PML, did not find an association between HHV-6 and PML (Quereda et al., 2000). Interactions between JCV and HHV-6 may play a part in PML. Increased expression of JCV as a result of HHV-6 superinfection of JCV-infected glial cells has been demonstrated (Yao et al., 2008). Although the data are limited, there is some evidence to suggest that HHV-6 infection or reactivation, in conjunction with JCV infection or reactivation, is associated with the demyelinative lesions of PML. Further research and antiviral treatment trials would be instructive in implicating a role for HHV-6 in PML.

Chronic fatigue syndrome CFS is a debilitating chronic illness characterized by severe fatigue and associated symptoms that last at least 6 months and cause significant functional impairment (Fukuda et al., 1994; Komaroff et al., 1996). Although the pathogenesis of this disease is unknown, abnormalities in the immune, endocrine, and nervous systems support an organic cause (Ortega-Hernandez and Shoenfeld, 2009; Yao et al., 2010). CFS often begins in the wake of infection with a variety of viral and non-viral microorganisms (Hickie et al., 2006), and there is evidence supporting HHV-6 as a trigger for this disease. The first large study exploring the association of HHV-6 with CFS involved a cohort of 259 patients experiencing a “CFS-like” illness (Buchwald et al., 1992). Active HHV-6 infection was identified in lymphocyte cultures from 70% of tested patients (n ¼ 113)

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versus 20% of healthy age- and gender-matched controls (n ¼ 40). HHV-6 replication was confirmed by assays using monoclonal antibodies specific for HHV-6 proteins and PCR assays for HHV-6 DNA in the lymphocyte cultures. This report had a number of limitations, as it lacked a clear CFS case definition, did not directly address whether HHV-6 produced the immunologic and neurologic dysfunction in affected patients, and enrolled patients from a relatively small geographic area. Mixed results are reported from subsequent research using serologic techniques that could not distinguish active from latent infection (Ablashi et al., 2000; Reeves et al., 2000; Hickie et al., 2006). Studies employing methods to identify HHV-6 replication (PCR of serum or plasma, IgM early antigen antibodies, and primary cell culture) have variably shown correlation (Patnaik et al., 1995; Secchiero et al., 1995; Ablashi et al., 2000; Nicolson et al., 2003; Komaroff, 2006) and lack of correlation (Reeves et al., 2000; Koelle et al., 2002) between active HHV-6 infection and CFS. These publications demonstrate that active HHV-6 infection is often observed with increased frequency in patients affected by CFS compared to healthy controls, although the findings are inconsistent. Given the neurologic and immunologic cell tropisms of HHV-6, it is a plausible causal agent of the biochemical and clinical abnormalities seen in this patient group. The propensity of HHV-6 infection to stimulate production and release of cytokines, both peripherally and in the CNS, may contribute to the symptomatology of this illness (Komaroff, 2006). HHV-6 reactivation may also be an incidental finding due to underlying immunologic dysfunction in CFS. Interestingly, some patients who have undergone HCT and develop blood HHV-6 reactivation suffer from cognitive dysfunction and fatigue similar to that reported by patients with CFS (Zerr et al., 2005a). Treatment studies using medications active against HHV-6 have resulted in symptom improvement and provide indirect evidence to implicate HHV-6 as one culprit etiology of CFS (Strayer et al., 1994; Kogelnik et al., 2006), although further research is needed.

Cognitive dysfunction after HCT HHV-6 reactivation following HCT and SOT is a common event (see section on “Spectrum of clinical disease: Immunocompromised host,” above). The clinical significance of this reactivation is not well understood, and there are no standardized guidelines for HHV-6 screening or treatment after transplantation. Several studies have suggested that HHV-6 reactivation in these patients can result in varying degrees of CNS dysfunction (Ljungman et al., 2000; Rogers et al., 2000a; Zerr et al., 2005a; Ogata et al., 2006; Yamane et al., 2007),

although these reports involved low patient numbers and lacked systematic assessment of CNS function. A recent prospective study in 315 patients undergoing allogeneic HCT found HHV-6 reactivation to be a strong predictor of CNS dysfunction (Zerr et al., 2011). HHV-6 was detected in plasma from 35% of patients within 84 days after HCT, and these individuals were significantly more likely to develop delirium or cognitive decline after controlling for other risk factors. These data suggest that HHV-6 may have a direct effect on the CNS, with blood reactivation serving as a marker of concurrent CNS disease. Since only 4 affected patients underwent one-time CSF sampling in this study (2 of whom had detectable HHV-6 DNA in the CSF) and no brain imaging or autopsy results were included in the analysis, the authors could not comment on a correlation between clinical symptoms and objective evidence of CNS disease. Worse patient outcomes after the occurrence of delirium in the posttransplantation period lends support to the clinical significance of HHV-6 reactivation as a contributor to delirium in this setting (Fann et al., 2007; Basinski et al., 2010). A causal relationship between blood HHV-6 reactivation and CNS dysfunction is strengthened by the finding that higher levels of HHV-6 viremia increased the association with cognitive decline (Zerr et al., 2011). A few small trials using ganciclovir to prevent HHV-6 reactivation in the early posttransplantation period had mixed efficacy in preventing a variety of associated complications, although cognitive dysfunction was not assessed (Rapaport et al., 2002; Tokimasa et al., 2002; Ogata and Kadota, 2008). Further studies are warranted to explore practical approaches to prevention and/or treatment of HHV-6 reactivation in immunosuppressed individuals and effects on neurologic outcomes.

Myelitis Myelitis, an inflammatory process of the spinal cord, is often caused by viral infection. A few case reports and case series have identified HHV-6A and B infections as likely etiologies of myelitis in both immunocompetent and immunosuppressed patients (Hill et al., 1994; Denes et al., 2004; Portolani et al., 2006; Mori et al., 2007, 2010; Pot et al., 2008; Troy et al., 2008). These associations were based on variable combinations of clinical, laboratory, and imaging studies. Myelitis as a complication of HHV-6 infection of the CNS appears to be a rare event and is not well established at this time.

CNS malignancy Since members of the herpesvirus family have been associated with malignancy (EBV, HHV-8), there has been interest in discovering a link between HHV-6 and neoplastic events. The neurotropism exhibited by HHV-6, along with its ability to establish lifelong latency in the

HUMAN HERPESVIRUS 6 AND THE NERVOUS SYSTEM CNS, makes it an attractive candidate agent in CNS neuro-oncogenesis. Saddawi-Konefka and Crawford (2010) comprehensively review the role of chronic viral infections in primary CNS malignancies and conclude that there is no definitive evidence of causality. A few studies employing HHV-6-specific PCR and other assays on primary brain tumor biopsy specimens compared to healthy control samples found a similar frequency of infection (Luppi et al., 1995; Chan et al., 1999; Cuomo et al., 2001; Neves et al., 2008), although two studies report an increased rate of HHV-6 detection in neoplastic tissue (Crawford et al., 2009b, c). Perhaps the strongest association between viral infection and CNS malignancy is for EBV and primary CNS lymphoma, typically in patients with HIV/AIDS (Gulley, 2001; Corti et al., 2004; Volpi, 2004). Studies focusing on detection of HHV-6 antigens or DNA in lymphoid lesional tissues from several lymphoproliferative disorders (Rojo et al., 1994; Luppi et al., 1998), as well as studies of immunocompetent and HIV/AIDS patients specifically with primary CNS lymphoma (Paulus et al., 1993; Cinque et al., 1996; Broccolo et al., 2000; Quereda et al., 2000), also argue against a major role for HHV-6 in the pathogenesis of primary brain tumors of neuroglial origin. Implicating chronic viral infections, and specifically HHV-6, as a cause of CNS malignancy by detection alone is controversial (Saddawi-Konefka and Crawford, 2010). Data interpretation is thwarted by variations in detection frequencies and methodologies, the role of the CNS as a reservoir for many latent neurotropic viruses in both normal and diseased states, and the widespread seropositivity for HHV-6 among the population. While direct evidence of HHV-6 as a mediator of neuro-oncogenesis is lacking, a model of neuro-oncomodulation has been proposed. Insight into the role of HHV-6 and other viral agents in CNS malignancies will be gained by cooperative use and validation of detection methods, as well as design and implementation of clinical trials targeting specific viral infections or modifying CNS neuroimmunity.

TREATMENT Acute infection with HHV-6 primarily occurs in immunocompetent children, resulting in mild and self-limited symptoms that generally do not require treatment. Infection or reactivation among immunocompromised individuals, however, may cause life-threatening complications and warrants aggressive management. It is possible that prophylaxis against, or early treatment of, HHV-6 after HCT or SOT will improve outcomes, as with CMV. Several antiviral agents demonstrate good in vitro and in vivo activity against HHV-6 (Table 16.5), including foscarnet, ganciclovir, and cidofovir (Burns and Sandford, 1990; Agut et al., 1991; Zerr et al.,

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2002a; De Bolle et al., 2005; Akhyani et al., 2006), but there have been no controlled trials to study these agents for HHV-6 therapy. Although the US Food and Drug Administration has not approved any antiviral drugs for the treatment of HHV-6 infections, the International Herpes Management Forum recommends foscarnet and ganciclovir, either alone or in combination, for treatment of HHV-6-related CNS illness (Dewhurst, 2004).

Ganciclovir and acyclovir Ganciclovir and acyclovir are acyclic nucleoside analogs. These chemicals are phosphorylated by a thymidine kinase specific to b-herpesviruses like HHV-6, creating a phosphorylated metabolite that inhibits viral DNA polymerase enzymatic function via competition for its natural substrate (dGTP) (Reardon and Spector, 1989). When incorporated into a growing DNA chain, these metabolites can effect chain termination. The in vitro activity of ganciclovir against HHV-6 is notably superior to acyclovir; acyclovir has poor efficacy for treatment of this virus (Yoshida et al., 1998; Manichanh et al., 2000). Both A and B species are equally sensitive to nucleoside compounds. Ganciclovir has a poor side-effect profile in many patients who would benefit from its use, as it can result in neutropenia and thrombocytopenia (McGavin and Goa, 2001). Its low oral bioavailability also mandates intravenous administration, although an oral prodrug, valganciclovir, offers similar blood levels (Brown et al., 1999). Plasma and CSF HHV-6 viral loads have been shown to decrease in vivo with ganciclovir treatment of patients with HHV-6-PALE (Zerr et al., 2002a; Ogata et al., 2006). Multiple studies support the clinical effectiveness of a 3–4-week course of ganciclovir for HHV-6-PALE in HCT or SOT recipients (Mookerjee and Vogelsang, 1997; Rieux et al., 1998; Johnston et al., 1999; Paterson et al., 1999; Yoshida et al., 2002), although approximately 50% of patients have persistent neurologic sequelae and up to 25% of cases do not respond to treatment (Tiacci et al., 2000; Rossi et al., 2001; Zerr, 2006a; Muta et al., 2009).

Foscarnet Foscarnet is a pyrophosphate analog with broad-spectrum activity against all herpesviruses (Wagstaff and Bryson, 1994; De Bolle et al., 2005). This drug directly targets the herpesvirus DNA polymerase by reversible binding near the pyrophosphate-binding site of the enzyme, which inhibits pyrophosphate release from amino acids and results in DNA chain termination. Notably, this drug does not require activation by cellular or viral enzymes, and its selectivity stems from a higher affinity for viral DNA polymerases than cellular DNA polymerases. In vitro studies have demonstrated excellent activity against

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Table 16.5 Antiviral agents active against human herpesvirus 6 (HHV-6) Antiviral agent

Efficacy*

Target, mechanism

Toxicity

Resistance{

Acyclovir{

þ

Renal

–

Ganciclovir{

þþ

Bone marrow

In vitro In vivo

Foscarnet

þþþ

Renal

–

Cidofovir{

þþþ

Renal

In vitro

Brincidofovir (CMX001, HDP-CDV){

þþþþ

Gastrointestinal

–

Cyclopropavir (ZSM-I-62){

þþþ

–

–

CMV423

þþþþ

Viral DNA polymerase Acyclic nucleoside analog Competes with dGTP ! chain termination Viral DNA polymerase Acyclic nucleoside analog Competes with dGTP ! chain termination Viral DNA polymerase Pyrophosphate analog Reversible binding ! chain termination Viral DNA polymerase Acyclic nucleotide phosphonate analog Competes with dCTP ! chain termination Viral DNA polymerase Cidofovir lipid ester analog Competes with dCTP ! chain termination Viral DNA polymerase Methylenecyclopropane nucleoside analog Competes with dGTP? ! chain termination Tyrosine kinase inhibitor? Non-nucleoside tetrahydroindolizine derivative Affects steps prior to DNA replication

–

–

*Loosely based on comparative studies in vitro. { Clinical or laboratory isolates with documented drug resistance. { Requires activation by viral or cellular enzymes.

HHV-6 and low concurrent cytotoxicity (Burns and Sandford, 1990; Reymen et al., 1995; Manichanh et al., 2000; De Clercq et al., 2001). Along with cidofovir, it has the best efficacy against HHV-6-infected astrocytes in vitro (Akhyani et al., 2006). Reversible dose-dependent nephrotoxicity is the primary side-effect. There are multiple reports showing both good (Bethge et al., 1999; Zerr et al., 2002a; Seeley et al., 2007) and limited or no (Tiacci et al., 2000; Rossi et al., 2001) responses to foscarnet use in transplant patients with HHV-6-PALE. No prospective trials have compared ganciclovir with foscarnet therapy for this purpose. Most large centers that care for patients with HHV-6-PALE use foscarnet for treatment in patients with normal renal function (estimated creatinine clearance >50 mL/min) because of a favorable side-effect profile compared to ganciclovir (Gewurz et al., 2008). Foscarnet should be started intravenously at a dosage of 60 mg/kg every 8 hours for 21–28 days with dose reduction in patients with renal insufficiency. Close monitoring of patients’ electrolytes and renal function while receiving foscarnet is essential. Treatment should always be guided by objective evidence of HHV-6 infection with CSF PCR testing if possible.

Cidofovir Cidofovir is an acyclic nucleotide phosphonate analog of deoxyCMP (De Bolle et al., 2005). This antiviral agent exhibits activity against a broad spectrum of DNA viruses, and it is often used to treat viruses resistant to other medications. Cidofovir requires phosphorylation by cellular enzymes for transformation into its active state. In this state, it acts as a competitive inhibitor of dCTP and an alternate substrate for the herpesvirus DNA polymerases, indirectly causing chain termination (Xiong et al., 1996). The selectivity of cidofovir is due to its higher affinity for viral DNA polymerases. This drug has strong activity against HHV-6 in vitro (Reymen et al., 1995). Along with foscarnet, it is the most active compound available for the treatment of HHV-6 infections (Yoshida et al., 1998; De Bolle et al., 2004a). However, due to the high risk of nephrotoxicity with this medication, as well as insufficient data regarding its capacity for CNS penetration, cidofovir is not currently recommended for the treatment of HHV-6-related CNS illness. One clinical report of the use of cidofovir in a patient with HHV-6 encephalomyelitis was interrupted due to drug toxicity (Denes et al., 2004). The patient

HUMAN HERPESVIRUS 6 AND THE NERVOUS SYSTEM 343 was subsequently treated with ganciclovir, which appResistance and new treatments eared to have superior efficacy and safety in this case. There are only two documented cases of HHV-6 resistance to antiviral drugs during active infection, both Prophylaxis of which were ganciclovir-resistant mutations that occurred after HCT and SOT in patients receiving therProphylaxis against HHV-6 infection or reactivation in apeutic doses of ganciclovir (Isegawa et al., 2009; populations at high risk for subsequent complications Baldwin, 2011). Experience with CMV prophylaxis and (i.e., immunocompromised patients) is under study. treatment may help predict development of HHV-6 Most patients who have undergone HCT already receive resistance if suppressive methods are instituted. Emeracyclovir or ganciclovir for HSV and/or CMV prophygence of CMV resistance is facilitated by drug exposure laxis, and there are trials demonstrating the efficacy of during active viral replication, after prolonged drug ganciclovir, but not acyclovir, in suppressing HHV-6 exposure, and when suboptimal drug doses are used reactivation (Wang et al., 1999b; Rapaport et al., 2002; (De Bolle et al., 2005). There is one report of a mutant Tokimasa et al., 2002). However, universal prophylaxis HHV-6 strain exhibiting cross-resistance to both ganciwith ganciclovir after HCT is not standard practice due clovir and cidofovir that emerged in vitro after serial to its bone marrow-suppressive effects, and the clinical passage under increasing ganciclovir pressure benefit of HHV-6 suppression is still unclear. (Manichanh et al., 2001). This strain carried mutations A study monitoring weekly plasma HHV-6 DNA resulting in amino acid substitutions in the HHV-6 levels in a post-HCT patient cohort showed no difference pU69 phosphotransferase and DNA pol regions, analoin mortality at 3 or 6 months among the 20 patients gous to the CMV UL97 and DNA pol regions that often treated with anti-HHV-6 agents, despite a significant develop mutations, resulting in CMV resistance. Similar decrease in HHV-6 viremia (Betts et al., 2011). This study HHV-6 mutations (without symptomatic HHV-6 related concluded that neither the occurrence of HHV-6 viredisease) were isolated in a patient with AIDS who mia, degree of viremia, nor use of antiviral drugs influreceived long-term ganciclovir therapy for CMV. Gancienced short-term survival after HCT. A study exploring clovir resistance from mutations in the HHV-6 pU69 the utility of pre-emptive ganciclovir use to prevent gene has since been replicated in vitro (Safronetz development of HHV-6-PALE in high-risk patients et al., 2003). Cidofovir-resistant mutants have also been (HHV-6 reactivation with  1  104 copies/mL plasma) selected in vitro due to a mutation in the U38 gene encodwas unsuccessful (Ogata and Kadota, 2008). In this ing the viral DNA polymerase (Bonnafous et al., 2008). experiment, 2 out of a cohort of 29 patients undergoing Emergence of HHV-6 resistance merits careful monHCT developed HHV-6-PALE, despite weekly moniitoring among patients maintained on long-term, lowtoring of HHV-6 plasma viral loads. Both patients had dose medications for prophylaxis of other herpesviruses. viral loads below the treatment threshold of  1  104 All available anti-HHV-6 drugs target the viral DNA copies/mL plasma prior to developing encephalitis, but polymerase, underscoring the importance of developing repeat testing after symptom onset revealed signifinew agents to target other steps in the HHV-6 lifecycle. cantly higher viral loads above the treatment threshold. A number of new medications for herpesviruses are in The dynamic kinetics of plasma HHV-6 viremia made various stages of development and testing, although prevention of HHV-6-PALE difficult. most appear to lack activity against HHV-6 (Coen and A subsequent study evaluated the safety and efficacy Schaffer, 2003). CMV423, a non-nucleoside tetrahyof pre-emptive treatment for HHV-6-PALE with foscardroindolizine derivative, is a new antiviral agent with net in 20 patients undergoing HCT (Ishiyama et al., 2011). potent and selective in vitro activity against the b-herpesTreatment was started for HHV-6 viral loads >500 copviruses, including HHV-6 (De Bolle et al., 2004a). ies/mL, as detected by thrice-weekly plasma PCR testing. It compared favorably to ganciclovir and foscarnet due Eight patients had HHV-6 viremia within 36 days after to its high activity and low cytotoxicity. CMV423 appears HCT, all of whom underwent UCBT. Mild and transient to exert its activity against HHV-6 through inhibition of a adverse events were associated with foscarnet in 7 of tyrosine kinase that plays an important role in early stages 8 treated patients, and 1 patient developed HHV-6-PALE of viral replication (following viral entry but preceding soon after starting foscarnet. HHV-6 reactivated as early viral DNA replication). Another study has shown a numas 10 days after HCT and often before neutrophil engraftber of methylenecyclopropane analogs of nucleosides to ment. In order to prevent HHV-6-PALE, perhaps highhave excellent activity against HHV-6, with minimal risk patients (e.g., patients undergoing UCBT) should cytotoxicity (Kern et al., 2005). Mechanism of action receive prophylactic foscarnet starting a week after studies in CMV suggested that these compounds are HCT and continued for a few weeks to prevent HHV-6 phosphorylated by the HHV-6 pUL97 phosphotransferreactivation. The efficacy of this approach is being examase to become potent inhibitors of viral DNA synthesis. ined in a prospective study.

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Finally, a study of oral lipid analogs of cidofovir and cyclic cidofovir revealed them to be considerably more active against HHV-6 and other herpesviruses in vitro than cidofovir (Williams-Aziz et al., 2005). Perhaps the most exciting of these drugs is brincidofovir (previously CMX001 and HDP-CDV), a pill taken twice weekly that has broad-spectrum antiviral activity against all five families of double-stranded DNA viruses that affect humans, higher in vitro antiviral activity than cidofovir, and a favorable side effect profile without evidence of kidney or bone marrow toxicity (Bonnafous et al., 2013; Marty et al., 2013). Further testing and discovery of therapies effective against HHV-6 with low toxicity are imperative for safe and viable treatment options of severe infections and the institution of trials exploring the role of HHV-6 in CNS diseases.

CONCLUSION HHV-6 is gaining recognition as a potential cause of a myriad of CNS diseases in immunocompetent, and especially immunosuppressed, patient populations. The widespread prevalence of HHV-6 in the CNS complicates our understanding of its role in neurologic disease, and there is significant controversy regarding whether viral infection and replication cause, or merely correlate with, the associated pathologies discussed in this chapter. Complex interactions between HHV-6 and the host immune system further confound attempts to implicate this virus in pathologic processes. Assigning causality of disease to infectious or non-infectious agents has plagued scientists for many decades. The Henle–Koch postulates, formulated in the

late 19th century, were the first criteria established to guide this challenging endeavor (Koch, 1890). These tenets have undergone many modifications since then to adapt to the changing environmental and scientific climate (Evans, 1976). Despite this, they continue to be an imperfect tool, and the scientific community often struggles to definitively connect pathogen with disease. This task is particularly difficult for HHV-6 due to limited animal models and its early and ubiquitous presence in both healthy and diseased patient specimens. Novel technologies that allow for greater sensitivity to detect viral agents, such as molecular amplification methods like PCR, may be more misleading than revealing (Madeley, 2008). Single tests of detection and quantitation provide only a snapshot of the evolution of a virus’ effects on a system after initial exposure and cannot reliably place the infection or disease process in time. Thus, distinguishing between stages of infection onset, viral persistence, or viral clearance is rendered difficult by limited sampling. Interpretation of such results necessitates longitudinal studies to develop an epidemiologic understanding of pathogens that links infection to illness in time. Ultimately, a more complete understanding of the significance of HHV-6 in its associated diseases will require large, multicenter prospective studies, welldefined disease criteria, a standardized HHV-6 assay capable of discriminating between species, negative and positive controls, and a blinded study design (Voumvourakis et al., 2010). Whether HHV-6 is a commensal pathogen, marker of immune dysregulation, trigger of an autoimmune response, or directly neurotoxic mediator of CNS disease will remain uncertain until more refined studies are performed.

EXECUTIVE SUMMARY Human herpesvirus 6 (HHV-6) biology HHV-6 is a b-herpesvirus discovered in 1986 that causes exanthem subitum HHV-6 has two closely related species, HHV-6A and B HHV-6 can infect many cell types and is neurotropic HHV-6 establishes latency after primary infection and reactivates under conditions of immunosuppression HHV-6 causes direct and indirect cytotoxicity, as well as complex effects on the immune system HHV-6 epidemiology Most HHV-6 infections occur in the first 2 years of life, and adult seropositivity is >95% (HHV-6B has greater prevalence) HHV-6 is detectable in glial cells throughout the brain in 32–85% of individuals HHV-6A appears to be more neurotropic and neurotoxic than HHV-6B HHV-6 detection Many methods have been used to demonstrate prior and active HHV-6 infection (Table 16.3) Lack of standardization of detection methods and application to many sample types hamper data comparisons PCR assays for HHV-6 DNA in plasma, serum, or CSF are the most practical techniques for identifying active infection

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HHV-6 spectrum of clinical disease HHV-6 causes a diverse array of diseases among immunocompetent and immunosuppressed individuals (Tables 16.1 and 16.2) Febrile seizures (FS) FS are associated with up to one-third of primary HHV-6 infections, although a higher incidence of HHV-6 infection and CNS involvement in children with FS has not been consistently demonstrated Mesial temporal lobe epilepsy (MTLE) Up to two-thirds of temporal lobectomy specimens show evidence of HHV-6B infection in patients with MTLE but not other types of epilepsy MTLE is associated with glutamate toxicity and astrocyte dysfunction, which can be seen with HHV-6 superinfection of astrocytes Multiple sclerosis (MS) HHV-6 antigen has been localized to MS plaques in a number of studies, but there is variability in the frequency of HHV-6 detection from plasma, serum, CSF, and tissue samples of MS patients versus controls using different techniques There appears to be a correlation between HHV-6 and MS, but it is unclear if HHV-6 has a role in causation or is a marker of immune dysfunction Encephalitis HHV-6-associated encephalitis can be fatal and primarily affects immunocompromised patients, although it also occurs in immunocompetent patients Up to 1.5% of patients undergoing allogeneic HCT develop encephalitis due to HHV-6 Confusion, anterograde amnesia, seizures, SIADH, increased CSF protein, and MRI abnormalities are common (Table 16.4) A positive CSF PCR test for HHV-6 in the appropriate clinical setting may be diagnostic after ruling out ciHHV-6 Treatment should be started early with ganciclovir or foscarnet for 21–28 days Progressive multifocal leukoencephalopathy (PML) Limited data have found colocalization of actively replicating HHV-6 in oligodendrocytes infected with JC virus from PML lesions but not in adjacent, uninfected cells Chronic fatigue syndrome (CFS) HHV-6 infection is sometimes observed with increased frequency in patients affected by CFS, but this may be due to immunologic dysfunction intrinsic to CFS and requires further study Cognitive dysfunction after HCT Peripheral HHV-6 reactivation early after HCT was associated with cognitive dysfunction in one prospective study and may prompt consideration of prophylactic treatment Myelitis Myelitis has been attributed to HHV-6 infection of the CNS in both immunocompetent and immunosuppressed patients, but it appears to be a rare event Primary CNS malignancy There is no evidence for HHV-6 as a causative agent of CNS malignancies, although it may participate in neuro-oncomodulation Treatment of HHV-6-associated diseases There are no controlled trials studying antiviral agents for the treatment of HHV-6-associated diseases Ganciclovir, foscarnet, and cidofovir are available medications with high in vitro efficacy against HHV-6 but significant sideeffects and limited success rates in the treatment of HHV-6-associated encephalitis (Table 16.5) Drug trials in HHV-6-associated diseases of the CNS will be important in understanding the significance of HHV-6 infection CNS, central nervous system; CSF, cerebrospinal fluid; HCT, hematopoietic cell transplantation; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; SIADH, syndrome of inappropriate antidiuretic hormone secretion.

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