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Human Herpesvirus 6

DAVID H. DOCKRELL, M.D.,* THOMAS F. SMITH, PH.D., AND CARLOS V. PAYA, M.D., PH.D.

Human herpesvirus (HHV) 6 is a ~-herpes, DNA virus.This virus shows closest homology with cytomegalovirusand HHV-7. Infection usually occurs in infants 6 to 24months of age, and primary infection may result inroseola. HHV-6 infection in infants is the commonestcause of fever-induced seizures. Infection in adults is seenprimarily in immunocompromised hosts with solid organtransplants or in those with human immunodeficiency vi­rus infection. The virus is capable of pronounced interac­tion in vitro with cytomegalovirus and human immuno­deficiency virus and induces immunosuppression andapoptosis. The importance of these interactions in vivonecessitates further investigation. HHV-6 infection may

The development of molecular-based techniques has ledto the study of the pathogenesis of three new members

of the human herpesvirus (HHV) family. These viruses,labeled HHV 6, 7, and 8 in order of their discovery, havereceived considerable attention during the past decade, anda substantial amount of information has been generatedconcerning their role in causing human disease.

HHV-6 was first isolated in 1986 by Salahuddin andcolleagues.' Its discovery resulted from attempts to isolatenovel viruses in patients with lymphoproliferative disease.The virus was isolated from lymphocytes obtained frompatients with various lymphoproliferative disorders by us­ing methods to activate and maintain lymphocytes in long­term culture. HHV-6 could be transmitted to phytohemag­glutinin-stimulated peripheral blood lymphocytes (PBL)but not to transformed cell lines. Analysis of infected cellsrevealed cytopathic changes, unique viral nucleic acid se­quences by in situ hybridization, and specific viral antigensby indirect immunofluorescence assay; morphologically,the virus resembled existing herpesviruses. Specific anti­body to HHV-6 was identified in all 6 index cases but wasdetected in only 4 of 220 healthy blood donors. Because

From the Division of Infectious Diseases and Internal Medicine(D.H.D., C.V.P.). Division of Clinical Microbiology (T.F.S., C.V.P.),and Division of Experimental Pathology and Laboratory Medicine(C.V.P.). Mayo Clinic Rochester, Rochester, Minnesota.

*Current address: University of Sheffield Medical School, Sheffield,United Kingdom.

Address reprint requests and correspondence to Dr. C. V. Paya,Division of Infectious Diseases, Mayo Clinic Rochester, 200 FirstStreet SW, Rochester, MN 55905.

Mayo Clin Proc 1999;74:163-170 163

contribute to the pathogenesis of multiple sclerosis. HHV­6 may be diagnosed by viral culture, serology, or poly­merase chain reaction.

Mayo Clin Proc 1999;74:163-170

AIDS = acquired immunodeficiency syndrome; CMV = cy­tomegalovirus; EBV =Epstein-Barr virus; EIA =enzyme im­munoassay; HIV = human immunodeficiency virus; HHV =human herpesvirus; IL = interleukin; LTR = long terminalrepeat; mRNA =messenger RNA; MS =multiple sclerosis;PBL = peripheral blood lymphocytes; PCR = polymerasechain reaction

the infected cells were identified as B lymphocytes, thevirus was labeled human B-celllymphotropic virus.

Despite the initial report, it has become apparent thatHHV-6 is, in reality, a T-celllymphotropic virus." Con­trary to the initial report of rare detection of antibody toHHV-6 in healthy blood donors, the virus is ubiquitous andfound worldwide.' Some investigators have suggested thatHHV-6 should be regarded as two separate viruses-HHV­6A and HHV-6B-despite the sharing of significant (95%)genomic identity; nevertheless, the two variants have dis­tinctive biologic and epidemiologic characteristics.'

BIOLOGY OF THE VIRUSStructure

HHV-6 is 160 to 200 nm in diameter, contains DNA thatis packaged in a nucleocapsid of 90 to 110 nm in diametercomposed of 162 capsomeres, and is surrounded by anenvelope 20 to 40 nm in thickness.' HHV-6 is a ~-herpes

virus, as are cytomegalovirus (CMV) and HHV-7.5 Theenvelope contains lipoprotein and the glycoproteins, whichare responsible for biologic functions such as binding to thehost cell receptors or acting as the targets for neutralizingantibody. The viral infectivity is inactivated by ether andlipid solvents; multiple freeze-thaw cycles, especially inthe absence of a protein-rich environment, also destroy thephysical integrity of herpesviruses.

GenomeThe viral genome is arranged as a linear segment of

double-stranded DNA and is approximately 160 kbp inlength." The HHV-6 genome consists of a single long

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164 Human Herpesvirus 6 Mayo ClinProc , February 1999, Vol 74

Table I.-Differences Between Human Herpesvirus 6A and 6B*

Characteristic

Growthproperties

Restrictionenzymepatterns

Antigenic profile

Cytopathogenicity

Disease association

HHV-6A

Peripheral blood lymphocytes,transformedT-cell lines

Distinct from HHV-6B

Reactionwith monoclonal antibodiesagainst gp82-105 or p180

Suppression of growthfactor-inducedbone marrow maturation is greaterwithHHV-6A

Primary disease is uncertain, possibledisease in immunocompromised hosts

HHV-68

Peripheral bloodlymphocytes only

Distinctfrom HHV-6A, sequence variationsin terminalrepeat regions determinestrains

No reactionwith monoclonal antibodiesagainst gp82-1 05 or p180,reaction withmonoclonal antibodies against pI01

Less virulent in cell culture

Roseola and reactivation disease inimmunocompromised hosts

*HHV = human herpesvirus.

unique sequence (D) that is flanked by identical directrepeat segments at each end (DRLand DR

R) , resulting in

the arrangement DRL-D-DRR

•i The herpesvirus clea vage­

package motifs , pa c-l and pa c-T; are arranged one at eachend in the direct repeat segments so that , during the processof viral replication when the two ends of the linear segmentof DNA unite , a unique junctional element containing pa c­2-pa c-l is produced. The guanine and cytosine content ofHHV-6 DNA is 43 to 44%, the lowest of any knownherpesvirus;' no hybridization occurs between the HHV-6genome and other previously described herpesviruses, ex­cept for a fragment of the CMV genome, which is homolo­gous with a subgenomic fragment of HHV -6. Furthercomparison of HHV -6 and CMV reveals that the CMVgenome is approximately 50% longer, is more complexwith several gene s lacking counterparts in HHV-6, andcontains a genomic segment that is colinear with a l15-kbfragment of HHV-6.8 The only other herpesvirus thatshares a similar genomic arrangement is HHV-7. Thegenome of the D] 102 strain, an HHV -6A variant, contains119 open reading frames encoding 102 separate proteins. 'The genes encoding the major capsid protein, viral poly­merase, glycoproteins gB, gH, and gl., and phosphopro­teins have been identified.' The protein virus shows close sthomology to CMV, with 67% prote in homology, and toHHV-7, which shows a 50 to 60% nucleotide identity withHHV-6.

Strain Variation and Cell TropismHHV-6 occurs as two variants, HHV-6A and HHV-6B,

which differ regarding the cell types that can be infected,genomic homology (90 to 96 %) antigen profile,epidemiologic features, and, possibly, cytopathic potential

(Table I) .4,9 The strain originally described by Salahuddinand associates,1 the GS strain, was an HHV -6A variant that ,like the Dll02 strain , has been described in irnmuno­compromised adults but not in those with roseola. Thestrain s that have been cultured from cases of roseola are allHHV-6B and include the prototype Z29 strain.

HHV-6 is highly tropic for CD4 T lymphocytes.Growth in primary lymphocytes necessitates stimulationwith phytohemagglutinin and growth in interleukin (IL)-2­containing media. 1 HHV -6, however, also infects CD8 Tlymphocytes, natural killer cells, macrophages, mega­karyocytes, epithelial cell s, and neural cells. 10,1I The CD4molecule does not seem to be essential for infection of CD4lymphocytes. Antibody to CD4 does not block infection,and HeLa cells expre ssing CD4 are not infected with HHV­6.12•13

Viral InteractionsAn important consideration with respect to the biology

of HHV-6 is its potential ability to alter the natural historyof other viral infections by its interaction with these vi­ruses. The interaction may be indirect (HHV-6-inducedimmunosuppression may predi spose to more severe dis­ease when infection occurs with another herpesvirus orhuman immunodeficiency virus [HIV] 14) or direct as oc­curs in coinfection (HHV-6 and a heterologous virus mayoccur in mixed infections in patients with central nervoussystem infection"). The theoretic basis for these interac­tions is derived from experiments in vitro. Sequence simi­larity has been noted between the gB peptide of CMV andHHV -6, but whether this finding has consequence for im­munity against these viruses is unknown. 16 A potentialmechanism of interaction between HHV-6 and CMV has

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Mayo Clin Proc, February 1999, Vol 74

been described for coinfections-the glycoprotein gHlgLhetero-oligomer, which is necessary for infectivity, mayrepresent a heterodimer resulting from a mixture of theCMV glycoproteins gH and gL and the correspondingHHV-6 glycoproteins.'? Therefore, coinfection could haveconsequences on reactivation and cell tropism. Furtherstudies are needed to explore this relationship and also thereports of increased replication of the Epstein-Barr virus(EBV) induced by HHV-6. 18

Similarly, HHV-6 may interact with HIV. A significantcorrelation exists between the frequency and extent ofHHV-6 DNA, as detected by the polymerase chain reaction(PCR) in peripheral blood leukocytes of HIV-seropositivepersons, and the absolute number of CD4 T lymphocytes. 19In vitro, HHV-6 is a potent transactivator of the long termi­nal repeat (LTR) of HIV, resulting in accelerated HIVreplication in vitro. The HHV-6 responsive elements havebeen mapped to the NF-KB- and Spl-binding sites of theHIV-LTR and function independently from the TAR ele­ment (responsive to the HIV transactivator tat), althoughthe addition of tat may be additive in the presence of HHV­6.20,21 Conversely, transfected tat leads to increased repli­cation of HHV _6.22 The pCD4I (U27) gene locus of HHV­6 encodes an early-late class phosphoprotein, whichtransactivates HIV-LTR. 23 Some investigators, however,have demonstrated that HHV-6 can inhibit HIV replicationin vitro under certain circumstances." A second source ofinteraction may be the ability of HHV-6 to induce CD4messenger RNA (mRNA) expression in cells such as y-8Tlymphocytes, CD8 T lymphocytes, and natural killercells,21,25.26 an outcome that could alter the range of cellsinfected by HIV. A further possible interaction could beindirect because of the cytokine profile produced in re­sponse to HHV-6 infection." The potential interactions ofHHV-6 with other herpesviruses and the manner in whichthese in vitro findings translate clinically are intriguingissues that merit further study.

IMMUNITYHumoral immunity occurs promptly after primary infec­tion. In infants with infection, neutralizing antibodies de­velop 3 to 7 days after the onset of fever.' During thesecond week after infection, IgM antibody peaks and per­sists for up to 2 months, whereas IgG production peaks at 2to 3 weeks and persists for life in 95% of adults.>" Therelationship of reactivation disease with conditions associ­ated with impaired cell-mediated immunity suggests thatcell-mediated immunity must also be important. Neutraliz­ing antibodies target various proteins, including linear andconformational epitopes of the glycoproteins. Reactivationof HHV-6 infection can also trigger an increase in specificantibody concentration."

Human Herpesvirus 6 165

HHV-6 has important effects on the immune system.Infection of PBL leads to decreased expression of IL-2mRNA after mitogenic stimulation, resulting in an anergicstate in vitro." Whether the alteration in mRNA levels isthe result of decreased production or accelerated degrada­tion is unknown. HHV-6 infection induces secretion oftumor necrosis factor from infected peripheral bloodmononuclear cell cultures," but they are cell-line specific,although alterations in other cytokine levels have beendescribed. In vitro, HHV-6 infection of transformed T-celllines results in death by apoptosis and enhanced suscepti­bility to Fas-mediated apoptosis," an effect that occurspredominantly in uninfected cells. This observation wassupported by the demonstration that cells undergoingapoptosis had none of the cytologic characteristics of di­rectly infected cells and that they did not stain positive withan anti-HHV-6 antibody. In addition, apoptosis was in­duced by a virion-free supernatant of HHV-6 infected cells.Therefore, induction of apoptosis in uninfected cells maybe one mechanism by which HHV-6 induces immuno­suppression. The combined effect of decreased IL-2levelsand enhanced susceptibility to apoptosis theoretically con­tributes to the clinical manifestation of HHV-6 infections,including lymphopenia and immunosuppression. The clin­ical relevance of these phenomena, however, necessitatesclarification.

EPIDEMIOLOGYGeneral Population

HHV-6 seropositivity occurs worldwide and is ex­tremely prevalent; most studies report rates higher than90% in children older than 2 years of age.' Specific mater­nal antibody protects newborns from HHV-6 infection, andgradual loss of this antibody results in a nadir value ofseropositivity of 10% at 6 months of age. Thereafter,primary HHV-6 infection leads to the development of anti­bodies, and seropositivity peaks at 2 years of age. Thisinformation correlates with the findings in the Rochester,New York, cohort of pediatric patients infected with HHV­6, in whom the peak age for virus isolation was 6 to 9months." The exact mode of transmission has not yet beendetermined, but, in view of the high rates of seropositivity,the virus must be easily transmitted to infants by closehousehold contacts. HHV-6 is shed in saliva and urine.These sources represent likely modes of transmission."The role of breast milk in transmission of HHV -6 infectionhas not been determined. Of interest, seroconversion hasbeen noted in formula-fed infants, a suggestion that breastmilk is not the exclusive means of transmission.'

Seropositivity may decrease with age," but some re­ports have failed to confirm this hypothesis. Whether theloss of seropositivity with age has clinical significance is

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166 Human Herpesvirus 6 Mayo Clin Proc, February 1999, Vol 74

Table 2.-Clinical Features of Human Herpesvirus 6*

Population

Pediatric

Adult

Bonemarrow transplantrecipient

Solidorgantransplantrecipient

HIV-seropositive

Symptomatic infections

Roseola infantum (exanthem subitum),rash withoutfever, nonspecific illnesses

Feverwith lymphadenopathy, hepatitis,encephalitis

Fever,rash, cytopenia, hepatitis,? pneumonitis, ? encephalitis

Fever,rash, cytopenia, ? pneumonitis,hepatitis, ? encephalitis

? Pneumonitis, ? encephalitis

Complications

Fever-induced seizures, encephalitis,acutedisseminated demyelination

? MS or otherdemyelinating conditions,? lymphoproliferative syndromes

? Increased severity of CMVdisease

? Increased severity of CMVdisease

? Increased HIV-viralload, ? role inCD4T-celldepletion, ? role in AIDSdementia complex

*AIDS =acquired immunodeficiency syndrome; CMV=cytomegalovirus; HIV=humanimmunodeficiency virus; MS=multiple sclerosis.

unclear, as is the role of reactivation in immunocompetentadults. Adults who have not previously experienced HHV­6 infection may have development of primary infection asadults, but this situation is rare.

Immunocompromised HostsImmunocompromised patients with solid organ trans­

plants are at increased risk of HHV-6 infection. Whetherthis risk results from reactivation of latent infection orsuperinfection is unclear. In addition, some patients whohave remained seronegative during adult life may experi­ence primary infection while they are immunocom­promised. Rates of HHV-6 infection vary after transplan­tation according to the type of solid organ transplant andthe method of detection of infection (viral culture, serol­ogy, or PCR). Reported rates of infection are as follows:24 to 31% for liver transplant recipients, 38 to 66% forrenal transplant recipients, 14% for heart transplant recipi­ents, and 38 to 60% for bone marrow transplant recipients(who have been more extensively studied after transplan­tation than the solid transplant groupS).1O,28,34-37 Trans­planted solid organs, blood transfusion, and reactivation ofendogenous virus by immunosuppression have been im­plicated as risk factors for posttransplantation infec­tion. IO,38,39 In HIV-infected patients, PCR detection ofHHV-6 DNA in CD4 T lymphocytes has been more usefulthan serology for the laboratory diagnosis of acute infec­tion. Its detection correlates with high CD4 T-lymphocytecounts."

CLINICAL FEATURESInfection in Infants

HHV-6 infection is often asymptomatic. Symptomaticdisease occurs predominantly after primary infection ininfants and after either primary or reactivation disease inimmunocompromised adults (Table 2). Most primary in-

fections with HHV-6 occur in children between 6 and 24months of age.' Although many primary infections may besubclinical, several patterns of clinical disease have beendescribed. Yamanishi and coworkers" demonstrated con­vincingly the etiologic link between HHV-6 infection andthe development of roseola infantum (exanthem subitum orsixth disease). Roseola is characterized by the rapid onsetof high fever lasting 3 to 5 days and by an erythematousmacular or macular-papular rash that occurs after the tem­perature normalizes. The spectrum of clinical disease ininfants is variable. A report from Rochester, New York,documented HHV-6 viremia in 34 of 243 acutely ill infants(14%) who were 2 years of age or younger and werehospitalized with febrile illness." These infants had highfever, irritability, and inflammation of the tympanic mem­branes. Of interest, only 3 of the 29 patients (10%) de­scribed had the typical rash associated with roseola. In afurther study, 1,653 infants or children younger than 3years of age were prospectively analyzed, and 160 (9.7%)had HHV-6 viremia and seroconversion." In addition tononspecific febrile illness and otitis, the most frequentclinical symptoms were those associated with upper respi­ratory tract or gastrointestinal involvement. In contrast,infants younger than 6 months of age in whom primaryHHV-6 developed had an illness characterized by lowerfevers, greater irritability, and more gastrointestinal symp­toms but a lower frequency of upper or lower respiratorytract disease. HHV-6 infection has been estimated to ac­count for 25% of hospital emergency department visits forinfants 6 to 12 months of age." Of note, of the 160 patientswith primary HHV-6, 21 (13%) had fever-induced sei­zures, which were often prolonged, and HHV-6 infectionaccounted for one-third of all fever-induced seizures inchildren 2 years of age or younger in one study." Otherneurologic features that are based on strong clinical evi­dence include a bulging fontanelle and meningoenceph-

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Mayo Clin Proc, February 1999, Vol 74

alitis. Encephalitis may be focal and may be associatedwith acute demyelination. In addition to fever withoutrash, HHV-6 infection may also develop with a rash as theonly clinical manifestation. HHV -6 infection has beenassociated with a fatal case of fulminant hepatitis occurringin a 3-month-old infant ."

Infection in AdultsEpidemiologic studies suggest that relatively few adults

are seronegative for antibodies to HHV-6; therefore, pri­mary infection in this population is rare. Immunocompe­tent adults or older children who are seronegative for HHV­6 may have development of an infectious mononucleosis­like illness with negative test results for EBV or CMV .43

These patients have fever , lymphadenopathy, and hepatitisor encephalitis. After an afebrile illness, they may havebilateral nontender cervical lymphadenopathy," and HHV­6 has been suggested to be a cause of Kikuchi 's lymph­adenitis, a type of necrotizing lymphadenitis." HHV-6 hasbeen linked to several pathologic conditions, includingEBV-negative Burkitt's lymphoma, follicular large celllymphoma, angioimmunoblastic lymphoma, Hodgkin'slymphoma, Sjogren's syndrome, sarcoidosis, and systemiclupus erythematosus, but whether the isolation of HHV-6from lymphocytes or the detection of antibodies to HHV-6in these conditions reflects an etiologic role for HHV-6 hasnot been determined (this topic has been previously re­viewed'). Similar considerations apply to the associationof HHV-6 with chronic fatigue syndrome, Guillain-Barresyndrome, or facial paralysis. In view of the neurotropismand propensity to cause neurologic disease, the associationof HHV-6 with multiple sclerosis (MS) is intriguing andhas been researched more than the other previously men­tioned possible neurologic associations in adults. In onestudy, the presence of HHV-6 nuclear staining by immuno­cytochemistry was seen in oligodendrocytes from patientswith MS but not in control subjects , and the staining waslocated around MS plaques." This finding has been furthersupported by the documentation of increased rates of IgMto HHV-6 and of serum detection of HHV-6 DNA inpatients with relapsing-remitting episodes of MS in con­trast to those with chronic, progressive MS or several con­trol groups."

Infection in Immunocompromised HostsHHV-6 infection has been reported with greater fre­

quency in immunocompromised adults-namely, solid or­gan transplant recipients and HIV-infected patients-thanin immunocompetent persons. HHV-6, like all herpesvi­ruses, is capable of reactivating from latency, and thusthese host populations are at particular risk of complica­tions from HHV-6.

Human Herpesvirus 6 167

The clinical feature s ofHHV-6 in solid organ transplantrecipients have recently been reviewed." In general , thepathogenic effects induced by HHV-6 after transplantationcan be divided into two groups : symptoms directly attrib­utable to HHV-6 infection and symptoms due to reactiva­tion of other herpesvirus, especially CMV, which mayresult from HHV-6 infection leading to reactivation of theother virus. Symptomatic disease is more likely to resultfrom primary infection with HHV-6 than from reactivation.In immunocompromised hosts, clinical consequences di­rectly attributable to HHV-6 infection include fever, leuko­penia, rash, encephalitis, and interstitial pneumonitis.P'v"In bone marrow and solid organ transplant recipients,HHV-6 viremia may induce bone marrow suppression.w"Furthermore, in vitro studies indicated inhibition ofcolony-forming unit assays in HHV-6, specifically leadingto suppression of leukocyte production. In bone marrowtransplant recipients and patients with the acquired immu­nodeficiency syndrome (AIDS), high levels of HHV-6DNA in blood are associated with interstitial pneumonitis,but causality has not been completely demonstrated.50

Other clinical features of HHV-6 in immunocompromisedhosts include rash and encephalitis.'? Besides the neuro­tropism of HHV-6 and the reported associations with MS,HHV-6 has been implicated in the pathogenesis of whitematter demyelination in AIDS dementia complex, with theidentification of active HHV-6 replication at sites of de­myelination." Causality, however, has yet to be clearlydemonstrated.

In patients with reactivation infections, HHV-6 infec­tion manifests with isolated fever more frequently thandoes organ-specific disease , but clinical symptoms are of­ten absent ; thus, the principal effect of HHV-6 infection inassociation with solid organ transplantation may resultfrom its potential to exacerbate CMV disease. The clinicalevidence for this outcome is that seroconversion to HHV-6or fourfold increases in HHV-6 antibody levels after trans­plantation are associated with CMV disease, and HHV-6infection is often symptomatic only when associated withCMV infection. P-" Further work is needed to determinewhether the interaction is induced at the level of viralreplication or whether infection with the two virusesmerely reflects reactivation of both viruses due to severeimmunosuppression.

Similarly, HHV-6 reactivation in HIV-infected patientsis frequently asymptomatic but may have indirect effectson HIV replication. HHV-6 up-regulates the expre ssion ofthe CD4 molecule and increases HIV replication. t-? HHV­6 is frequently detected in HIV-infected patients ; it hasbeen etiologically linked with several clinical syndromes,but its most important effects could be due to its interactionwith HIV, through its role as an immunomodulator or

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168 Human Herpesvirus 6

Test

Table3.-Diagnostic Tests for IdentifyingHuman Herpesvirus 6*

Options

Mayo Clin Proc, February 1999, Vol 74

CultureSerology

Polymerase chainreactionImmunohistochemistry

*HHV =human herpesvirus.

Standard cell culture, shellvialtechniqueIndirect immunofluorescence assay, anticomplement

immunofluorescence assay, enzyme immunoassayCellular sample, acellular sample, semiquantitativeMonoclonal antibody to plOl (HHV-6B), monoclonal

antibody to gp82(HHV-6A)

modifier of HIV replication, and thus enhancement ofHIV-mediated CD4 T-cell depletion (this issue has beenpreviously reviewed"),

DIAGNOSISCulture

Viral culture is the gold standard for detecting HHV-6 inperipheral blood mononuclear cells (Table 3). Peripheralblood mononuclear cells are purified and cocultured withPBL, with cord blood lymphocytes, or, alternatively, byprimary cell cultures in RPMI-1640 medium.' Freshlyprocessed cells from blood specimens should always beused because the virus is labile after freeze-thaw cycles.Inoculated cell cultures are then examined at 3- to 4-dayintervals, beginning 5 to 7 days after infection, and ana­lyzed for specific cytopathic effects. Immunologic confir­mation of virus-infected cells should be ascertained byusing indirect immunofluorescence or anticomplement im­munofluorescence tests. The specificity of these tech­niques should be confirmed by using antibodies againstother herpesvirus-infected cells. Isolation by viral culturenecessitates 5 to 21 days (median, 11); an adaptation of thetechnique facilitates rapid detection of virus by the shellvial assay in 1 to 3 days. 10 The technique involves centrifu­gation of the specimen to facilitate inoculation of host cellsand subsequent immunologic detection in early antigens ofvirus-infected cells. A sensitivity of 86% and a specificityof 100% have been reported for this technique for speci­mens from solid organ transplant recipients.

SerologyPrimary infection with HHV-6 in immunocompetent

hosts can be demonstrated serologically by seroconversionof IgG from negative to positive or the presence of IgM toHHV-6. A fourfold increase in IgG antibody detected byimmunofluorescence or a 1.6-fold increase in antibodymeasured by enzyme immunoassay (EIA) indicates recentinfection; however, distinguishing between primary infec­tion and reactivation under these circumstances may bedifficult. Similarly, the clinical significance of IgM detec-

tion in adults is less clear because IgM may be produced inboth primary and reactivation infections." The indirectimmunofluorescence assay is a sensitive method of anti­body detection, and a titer of 1:10 or greater is regarded aspositive, especially when diffuse fluorescence of enlargedcells is noted.

The anticomplement immunofluorescence assay is analternative method. Its potential advantages are that it canreduce nonspecific results due to binding of antibodies toFe receptors on the surface of cells and increase the signalfrom serum with positive results. With this procedure,guinea pig complement is allowed to react with specificantigen-antibody complexes, followed by the addition of afluorescein isothiocyanate-conjugated antibody to the C3component of complement. Nonspecific reactions areeliminated because C3 reacts only with immune com­plexes and not with IgG attached to Fc receptors. Thespecificity of the technique has been illustrated by its abil­ity to detect the same percentage of infected cells as elec­tron microscopy. 53

With the aforementioned assays, the slides must be readby a technologist experienced with immunofluorescenceassays. Because review is operator dependent, results maylack objectivity. In general, EIA techniques are moreeasily quantified and less subjective. An EIA protocolhas been developed for the laboratory diagnosis of HHV­6 infection. As originally described, the assay is specific,with no competitive cross-reactions with other herpes­viruses and loss of 90% of the binding activity by adsorp­tion with soluble HHV-6 antigen." The difficulty withthis assay has been that the results are distributed withina normal distribution curve without a bimodal distribu­tion representing positive and negative results. Establish­ing a cutoff value to define positive results is thereforedifficult.

Polymerase Chain ReactionPCR can be performed on either cellular or acellular

specimens to detect HHV-6 target DNA. If peripheralblood mononuclear cells are used, distinguishing active

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Mayo Clin Proc, February 1999,Vol 74

from latent infection is difficult, and ideally a quantitativetechnique for determining the copy number (viral load) ofthe virus should be used; however, these techniques are stillinvestigational.P Some investigators have suggested thatthe use of acellular samples will be more helpful in distin­guishing between these two situations, particularly inimmunocompromised hosts.'? Secchiero and colleagues"described a sensitive technique for the detection of HHV-6in serum. Two sets of nested primers are used: the firstderived from the major capsid protein gene and the secondfrom the putative large tegument protein. After HHV-6DNA is amplified with the outer primer pair, an aliquot ofthe PCR product is then used as a target to generate anamplicon internal to the original product by using a secondprimer pair. Amplified DNA is visualized by performingelectrophoresis with use of a 2% agarose gel and stainingwith ethidium bromide. The PCR procedure described issensitive for several HHV -6 strains and does not result in apositive signal from cells infected with other herpesviruses,including HHV-7. Healthy adults have no detectable viralDNA, but children with roseola and a variety ofimmunocomprornised patients yield positive PCR results.The technique facilitates semiquantification and straincharacterization by using a primer set mapping to a poly­morphic region of DNA.

ImmunohistochemistryImmunohistochemical tests can be performed on forma­

lin-fixed, paraffin-embedded tissues with use of murinemonoclonal antibodies against the structural protein plOlof HHV-6B or against glycoprotein gp82 of HHV-6A. 1O

Only cells with active infection, in contrast to latent infec­tion, stain positively with these antibodies. This techniquefacilitates identification of HHV-6 in a biopsy or cytologicspecimen within 1 to 3 days after collection. The techniqueis easily performed in any pathology department.'?

SummaryHHV-6 infection can be diagnosed with any of the

previously mentioned techniques. Monoclonal antibodiesare commercially available for use in these applications.The application of these reagents to viral culture andserologic diagnosis is operator dependent, however, andexperience with these procedures facilitates interpreta­tion. PCR-based techniques are additional valuable re­search tools. Primary infection during infancy is a clinicaldiagnosis that can be confirmed by serology, if neces­sary. In immunocompromised hosts, HHV-6 infection isbest documented by one of the other methods described.PCR or rapid shell vial are suitable tests with serumsamples, whereas immunostaining is beneficial with bi­opsy specimens.

Human Herpesvirus 6 169

THERAPYBecause HHV-6 is similar to CMV, the spectrum ofantivirals with activity against this virus is similar.'? Invitro, ganciclovir and foscarnet have good activity, but thevirus is relatively resistant to acyclovir. Nevertheless, therole of antiviral therapy for HHV-6 infection has yet to bedetermined. In transplant recipients, investigators havesuggested that those with evidence of hepatitis,pneumonitis, or encephalitis and positive results of a diag­nostic test for HHV-6 should receive therapy;'? however,HHV-6 is often encountered in association with a secondpathogen or with graft rejection, and thus management ofthese cases is complex. Therapy is usually unnecessary foruncomplicated primary infections in immunocompetentpatients.

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