INVITED REVIEW

Blue Moon Neurovirology: The Merits of Studying RareCNS Diseases of Viral Origin

Lauren A. O'Donnell & Glenn F. Rall

Received: 23 January 2010 /Accepted: 5 March 2010 /Published online: 24 April 2010# Springer Science+Business Media, LLC 2010

Abstract While measles virus (MV) continues to have asignificant impact on human health, causing 150,000–200,000 deaths worldwide each year, the number offatalities that can be attributed to MV-triggered centralnervous system (CNS) diseases are on the order of a fewhundred individuals annually (World Health Organization2009). Despite this modest impact, substantial effort hasbeen expended to understand the basis of measles-triggered neuropathogenesis. What can be gained bystudying such a rare condition? Simply stated, the wealthof studies in this field have revealed core principles thatare relevant to multiple neurotropic pathogens, and thatinform the broader field of viral pathogenesis. In recentyears, the emergence of powerful in vitro systems, novelanimal models, and reverse genetics has enabled insightsinto the basis of MV persistence, the complexity of MVinteractions with neurons and the immune system, and therole of immune and CNS development in virus-triggereddisease. In this review, we highlight some key advances,link relevant measles-based studies to the broader dis-ciplines of neurovirology and viral pathogenesis, andpropose future areas of study for the field of measles-mediated neurological disease.

Keywords measles virus . neuron . SSPE . CNS infection

AbbreviationsCDV canine distemper virusCNS central nervous systemCSF cerebrospinal fluidF fusion proteinGFP green fluorescent proteinH hemagglutinin proteinIFNγ interferon gammaIL interleukinISG interferon-stimulated geneL RNA-dependent, RNA polymerase proteinLCMV lymphocytic choriomeningitis virusM matrix proteinMHV mouse hepatitis virusMIBE measles inclusion body encephalopathyMV measles virusN nucleoproteinNK-1 neurokinin-1NSE neuron-specific enolaseP phosphoproteinPIE post-infectious encephalomyelitisRNP ribonucleoproteinSLAM signaling lymphocyte activation moleculeSSPE subacute sclerosing panencephalitisSTAT signal transducer and activation of transcriptionTh T helperYAC yeast artificial chromosome

A current view of the impact of measles virus on humanhealth

Before the introduction of an effective live-virus vaccine in1963, measles virus (MV) was a major cause of infant

L. A. O'Donnell :G. F. Rall (*)Program in Immune Cell Development and Host Defense,Fox Chase Cancer Center,333 Cottman Avenue,Philadelphia, PA 19111, USAe-mail: glenn.rall@fccc.edu

J Neuroimmune Pharmacol (2010) 5:443–455DOI 10.1007/s11481-010-9200-4

mortality throughout the world. The vaccine has beenremarkably successful in preventing acute infections,resulting in an estimated 95% decrease in the incidence ofinfection in the US within 5 years of its introduction. Whilethe worldwide incidence of infection continues to decline,largely due to improving vaccine coverage, the WorldHealth Organization (2009) estimates that 150,000–200,000 people still die each year of complications fromthis infection.

As with most human pathogens, healthcare issues andthe challenges to vaccination and pathogen eradicationdiffer depending on what part of the world is beingconsidered. In resource-poor regions, MV remains theleading cause of vaccine-preventable deaths in children(Centers for Disease Control and Prevention (CDC) 2006;Moss and Griffin 2006). The substantial impact of MV indeveloping countries can be attributed to multiple factors,including: lack of vaccination, inoculation with an uninten-tionally inactivated vaccine (due to lapses in the “coldchain” necessary to keep the attenuated virus efficacious),or vaccination prior to the waning of maternal antibodies.These problems, relevant as much to the public healthinfrastructure of the impacted countries as they are to viralbiology, are extraordinarily challenging to address and,thus, regrettably, the morbidity and mortality toll of MV isunlikely to substantively improve until concomitantchanges in public health policy occur.

In contrast, resource-rich countries such as the UnitedStates typically have high vaccination coverage (>90%)and even those few individuals who are not vaccinatedbenefit from herd immunity that limits access of thevirus into the community. However, countries withhistorically high vaccination rates are facing uniqueproblems as well. While many perceive measles to be adisease of the past, the recent and alarming wave ofmeasles infections in the US and Europe have resurrectedvalid fears about MV susceptibility in typically well-protected communities (Editorial Team 2008). In 2008,the CDC reported 131 cases of MV in the US, the highestsince 1996 (Centers for Disease Control and Prevention(CDC) 2008). Virtually all of the recent acute MV caseshave occurred in children who were not vaccinated; ofthese, many were not vaccinated due to parental concernsabout the perceived association of vaccines (measles inparticular) with childhood autism. This controversy wasinitially fueled by a study published in Lancet in 1998 thatreported an association of autism diagnosis with thepresence of MV RNA in the gut (Wakefield et al. 1998).Despite its eventual retraction in 2010 (by the journal)and abundant studies that refuted any associationbetween vaccination and autism, many families stillharbor concerns. Though parents must make the choicesthey deem most appropriate for their children, the

decision to delay or decline vaccination has imperiledthe critical threshold needed for effective herd immuni-ty, and—predictably—outbreaks of MV have occurred inmany communities. At the very least, this has been a tellingexample that complete eradication of a pathogen requiresboth efficacious interventions (such as vaccines) as well asa community that is willing to use them. In that respect,pathogen eradication is as much a matter of marketing,public discourse, and politics, as it is development of aprotective vaccine.

Measles virus biology and cellular receptors

A number of comprehensive reviews (Moss and Griffin2006; Rima and Duprex 2009) have recently beenpublished concerning the replication and pathogenesis ofmeasles; thus, the basics of measles biology and diseasewill be reviewed only briefly here. MV is a prototypicparamyxovirus, consisting of an enveloped, negative-strand, ∼16-kb RNA genome encoding eight proteins.These proteins include replication factors [RNA depen-dent, RNA polymerase (L) and phosphoprotein (P)],structural proteins [nucleoprotein (N), matrix (M), hem-agglutinin (H), and fusion (F)], and two accessoryproteins that play a role in pathogenesis and replication(C and V; Poole et al. 2002; Rodriguez et al. 2003a). Theviral ribonucleoprotein (RNP), formed within the cyto-plasm of infected cells, consists of the viral genomecomplexed with N, P, and L. The classical view of MVspread is that, as the virus buds from the cell, it acquiresits envelope, including H and F from the plasmamembrane. The M protein is required to correctlytransport H and F to the plasma membrane (Naim et al.2000) and may also play a key role in bringing the RNPinto physical approximation with the envelope proteins(Manie et al. 2000; Vincent et al. 1999). Infection of newcells is initiated when MV-H on the virion (or the infectedcell) attaches to a cellular receptor on a susceptible cell.This, then, triggers exposure of a fusogenic domain onMV-F, resulting in fusion between the virus and host cellmembranes or between an infected cell and an adjacentuninfected cell.

Mice do not possess functional MV receptor homo-logues, and thus are not susceptible to infection by wild-type or vaccine strains. With the identification of twohuman MV receptors: CD46 in 1993 (Dorig et al. 1993;Naniche et al. 1993) and SLAM/CD150 in 2000 (Tatsuoand Yanagi 2000), it became possible to develop transgenicmice expressing these receptors, with the aim of establish-ing permissive mouse models (reviewed in Manchester andRall 2001). As described in greater detail below, suchmodels have been invaluable in defining key elements of

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replication and pathogenesis for this otherwise human-restricted pathogen.

Measles-induced pathogenesis

Following exposure of a naïve individual to measles by anaerosol route (e.g., droplets from a sneeze), there is a fairlylong incubation period (5–10 days) prior to the appearanceof any symptoms, during which time the individual is ableto spread the infection to others. This period culminates insigns of sickness, including fever, cough, and congestion,that are similar to other respiratory infections. However,unlike most respiratory-restricted infections, other symp-toms emerge during MV infection, including a characteris-tic maculopapular rash and the early appearance Koplik'sspots in the oral mucosa. Resolution for most infectedindividuals occurs without consequence, usually within10 days after the first signs of sickness. Immunity to MV isthought to be life-long.

Two other sequelae associated with MV exposure areimmunosuppression and central nervous system (CNS)disease. Of these two, the vast majority of MV-related deathsare attributable to transient immunosuppression, which allowsfor rapid colonization and unrestricted growth of opportunisticinfections. In regions of the world where sanitation is absentor inadequate, unchecked growth of bacterial or parasiticpathogens can precipitate illnesses such as diarrhea that,without medical intervention, can be fatal. Additionally,pneumonia is a common and equally serious consequence ofinfection. The observation that MV infection impinges onimmunity to other pathogens has been appreciated since thebeginning of the twentieth century, but only in the past twodecades have we begun to discern the mechanism by whichthis occurs (comprehensively reviewed recently by Schneider-Schaulies and Schneider-Schaulies 2009). Multiple, non-mutually exclusive hypotheses exist to explain how thisvirus (which maximally infects 2% of T lymphocytes) canresult in such widespread immunosuppression. These well-supported theories include: MV blunting of interleukin 12production by professional antigen-presenting cells, skewingthe subsequent T cell response away from the desired Thelper 1 (Th1) type toward the less useful Th2 type (Hahm etal. 2007; Karp et al. 1996; Yu et al. 2008); sequestration ofkey interferon signaling molecules in the cytoplasm, pre-venting them from entering the nucleus and binding theirpromoter targets (Palosaari et al. 2003; Ramachandran et al.2008); viral protein-mediated inhibition of naïve lymphocyteproliferation (Schlender et al. 1996; Schnorr et al. 1997); andinfection of bone marrow stromal cells that disruptsmaturation of “prelymphocytes” (Manchester et al. 2002).In humans, MV infection is associated with a marked drop inCD4+ and CD8+ T cells, potentially due to the inhibition of T

cell proliferation and cell cycle progression (Naniche et al.1999; Niewiesk et al. 1999; Schnorr et al. 1997), that isultimately recovered after the primary infection (Ryon et al.2002) and a dampened response of peripheral bloodlymphocytes to antigenic stimulation ex vivo (Borrow andOldstone 1995; Hirsch et al. 1984). Finally, a possible rolefor regulatory T cells has been proposed based upon a mousemodel of MV infection, which demonstrated an increasedfrequency of these cells in the periphery and the CNS,accompanied by a decrease in the hypersensitivity response(Sellin et al. 2009). It is likely that many, if not all, of thesestrategies are operative, underscoring the myriad ways bywhich this deceptively simple virus can frustrate the mamma-lian immune response to gain a replicative advantage.

Among the survivors of measles infection, a smallpercentage of individuals develop neurological sequelae thatcan lead to mortality weeks to months to years after the initialexposure. Typically, there are three diseases that are attribut-able to MV infection in the CNS that differ in their timing,frequency, and background of the infected host. Post-infectious encephalomyelitis (PIE) or acute disseminatedencephalomyelitis occurs in approximately one of 1,000measles cases and typically affects children and adolescents(Miller 1964). Symptoms of PIE include seizures, motor andsensory defects, and ataxia, typically begin 5–14 days afterthe primary MV infection has resolved, though they can bedelayed weeks to months after the primary infection. PIE ischaracterized by demyelination, with or without hemorrhag-ing and perivascular macrophage infiltration. There is littleevidence for MV infection in the brain during PIE, as MVantigen and RNA are undetectable by immunohistochemistryor in situ hybridization, respectively (Gendelman et al. 1984;Moench et al. 1988; Norrby and Kristensson 1997). PIE maybe an autoimmune disease, as patients have elevated immuneresponses to myelin basic protein (Gendelman et al. 1984;Johnson et al. 1984). PIE has a mortality rate of approxi-mately 25%, with survivors demonstrating persistent neuro-logical deficits (Chardos et al. 2003; Schwarz et al. 2001).Currently, there is no effective treatment.

Measles inclusion body encephalitis (MIBE) is a rareCNS complication following MV infection in immunocom-promised patients including those receiving immunosup-pressive drugs (Colamaria et al. 1989; Johnson 1998; Poonet al. 1998). Neurological symptoms, including seizures,motor deficits, and cognitive changes, appear 3–6 monthsafter primary MV infection (Johnson 1998; Perry andHalsey 2004). MIBE is characterized by the presence ofinclusion bodies within neurons, astrocytes, and oligoden-drocytes, with accompanying neuronal loss. Both MVantigen and RNA are detectable within brain samples, andvirus can be directly isolated from the brains of MIBEpatients (Baczko et al. 1988; Johnson 1998). However, noapparent inflammation is present and there is an absence of

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neutralizing antibodies in the serum and cerebrospinal fluid(CSF; Rima & Duprex 2005; Weissbrich and terMeulen2003). Death occurs in approximately 80% of MIBE caseswithin days to weeks of the initial neurological symptoms.

Subacute sclerosing panencephalitis (SSPE) is a slow,progressive neurological disease that appears a mean timeof 6–10 years after primary MV infection (Dubois-Dalcq etal. 1976; Modlin et al. 1977). SSPE typically develops inpatients under 20 years of age, though adult onset ispossible in rare cases (Singer et al. 1997). Symptoms ofSSPE commonly begin with personality changes and subtlecognitive losses before progressing to myoclonus, seizures,dementia, coma, and death over a period of weeks to yearsafter the initial onset of neurological symptoms. Unlike PIEand MIBE, SSPE is associated with extremely elevatedlevels of measles-specific antibodies in the CSF and serum(Ebinger and Matthyssens 1971). Neurons and oligoden-drocytes are the main target cell for MV in SSPE brains,though MV infection of astrocytes, endothelial cells, andinfiltrating lymphocytes has been noted (Allen et al. 1996;Kirk et al. 1991). Importantly, infectious MV cannot berecovered from SSPE tissues, implicating cellular or viralchanges that affect the viral life cycle. While MV RNA ispresent in SSPE brains, the mechanism for SSPE patho-genesis is unknown, though SSPE is marked by neuro-degeneration, astrogliosis, and markers for oxidative stressin the brain (Aydin et al. 2006). Treatments such asribavirin and interferon have produced conflicting resultsin SSPE patients, and there is currently no standardtreatment protocol for SSPE other than supportive care.

Viruses in the brain: what we know from clinical studies

How common are neuropathological sequelae for otherviruses that can target the CNS? While death due to virus-induced neuropathology is uncommon, many human virusesbesides MV have the potential to infect CNS parenchymalcells, including polio, influenza, mumps, rabies, West Nile,and some herpes viruses. As with measles, in those rare caseswhen acute, virally mediated CNS disease occurs, theprognoses are almost always poor. The severity of thesediseases is often due to inflammation, resulting in encephalitisand/or meningitis (reviewed in Rall 1998). Perhaps tomoderate these potentially deleterious effects of robustimmunity, the CNS has safeguards that collectively restrictboth the access and function of lymphocytes within theparenchyma (once called “immune privilege,” but nowrecognized as not simply “less” immunity, but rather as adistinct way to regulate immune responses in this criticaltissue). These anatomical and biochemical properties includethe blood–brain barrier, the absence of lymphatic drainage,the paucity of major histocompatibility complex class I

expression on most neurons, and elevated levels of immu-nosuppressive molecules such as gangliosides (reviewed inRall and Oldstone 1997). Together, these CNS featuresrestrict immune cell access into the CNS under normalconditions and may favor noncytolytic clearance strategiesduring pathogenic challenge (Binder and Griffin 2001; Parraet al. 1999; Patterson et al. 2002).

Fortunately, for most individuals infected with potentiallyneurotropic viruses, the infection is resolved before neuro-pathogenesis can occur. This may be attributable to resolutionof the infection before viral access to the CNS can beachieved. However, some evidence suggests that this simpleexplanation may not uniformly be the case. A study of tissuespecimens from aged individuals who died of nonviral (andnon-CNS) causes revealed that a large proportion of brainbiopsies contained MV RNA (Katayama et al. 1995),implying that —at least for MV— asymptomatic, quiescentinfections can be established in the CNS. While these studiesrequire validation with more sensitive methods that are nowavailable, they imply that viral neuroinvasion and neuro-pathogenesis are not inexorably linked, and the goal of thehost immune response may not be to clear the infectionaltogether, but rather to keep the persisting virus fromreactivating or spreading.

In addition to acute disease, neurotropic viruses can alsocontribute to chronic CNS diseases, similar to the long-lasting MV CNS diseases described above. Examplesinclude: spongiform encephalopathies caused by somelentiviruses, chronic neurodegeneration following Bornadisease virus infection, post-influenza encephalitis, andmumps meningoencephalitis (Johnson 1998). In addition,some have speculated that chronic CNS diseases ofunknown etiology, including schizophrenia, amyotrophiclateral sclerosis, and multiple sclerosis may have a viralcomponent or trigger (Berger et al. 2000; de la Torre et al.1996; Johnson 1998). While a formal association betweenthese illnesses and a viral agent requires further study, it iscertain that we do not yet fully appreciate the degree towhich the CNS is influenced by persistent viral infections.

The issue: does the small number of cases of CNSsequelae following MV infection justify efforts to studythese diseases?

While CNS diseases due to MV infection are almostunilaterally fatal, they are also notably rare, likely affectingfewer than one out of 10,000 acutely infected persons. How-ever, these estimations almost surely under-represent theneurological impact ofMV, since the latency between primaryinfection and CNS disease is so long and records are often notsufficiently detailed to connect CNS disease with a prior MVexposure. Moreover, individuals who may have eventually

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developed CNS sequelae may die earlier of other causes,including malnutrition or other infectious agents (reviewed inRall 2003). Even so, in weighing the impact of MVon humanhealth, these CNS diseases represent a small minority of thetotal MV burden. Why, then, is substantial effort invested instudying them and the basics of MV growth in the brain?

The point of view of this review is that the study of rarediseases can provide fundamental insights that may be morebroadly applicable to human disease. In particular, theestablishment of animal models and cell culture approachesto explore virus–neuron and virus–immune response inter-actions has revolutionized the study of MV biology within thebrain. We will focus on three areas of progress in theremainder of this review. First, the unique viral life cycleadopted by MV in CNS neurons of mice (and potentially, ofaffected patients) offers a window into how viruses adjusttheir replication and spread within cells in the brain, perhapsleading to evasion of the immune response. Secondly, it isclear from MIBE patients and animal models that acompetent, mature immune response is needed to controlMV in the brain. What is surprising is the widespreadutilization of noncytopathic strategies of viral control, chieflythrough the use of interferon gamma (IFNγ). Finally, MV isoverwhelmingly an infection of children; and animal modelsof MV infection mirror the age-dependent susceptibility. Bystudying MV in the brain, we are able to ask questions aboutinteractions between the immature immune system and adeveloping brain and to examine why viral clearance failspredominately in children.

Basic principle 1: the viral life cycle is not monolithicand cell-specific influences on the virus can affectpathogenesis

Human studies

The rarity of deaths ascribed to MV, and the dauntingchallenge of isolating brain tissues from these individualsof sufficient quality for subsequent analysis, continue topose challenges to uncovering the basis of MV-mediatedneuropathology. Of the small number of tissues that havebeen available, a few major points have emerged. First, thebrains of SSPE patients show dramatic and extensivepathology, sometimes described in the clinical literature as“decorticated” (Anlar 1997). Blood–brain barrier perme-ability breaches, glial activation, inflammation, and cellulardamage are all noted as pathologic hallmarks, thoughunraveling which of these neuropathological events areinitiating (that is, directly attributable to viral infection orimmunopahology) and which are secondary to the primarylesions is not possible to ascertain from these end-stageclinical specimens. A further challenge is deducing the state

of the virus in these brains over the long period betweeninitial infection and subsequent death. Is the virus quiescentfor years, at which point some reactivation event (similar,perhaps, to reactivation of a latent herpes virus infection)triggers rapid replication and precipitous disease, similar toreactivation of a latent herpesvirus infection? Or, is thevirus replicating at a slow but persistent rate, and only oncea particular brain region or some threshold number of cellsare infected do symptoms appear? While we do not yetknow the answer, either scenario (MV latency or glacialreplication rate) would suggest a major difference from thecanonical view of how this virus replicates.

One intriguing observation obtained from human studieswas the frequency of clustered mutations in MV genomesthat were sequenced from brain specimens. Extensive pointmutations, affecting as much as 2% of the genome(Cattaneo et al. 1988) and occurring in clusters, particularlyin the F and M genes, were consistently identified in SSPEand MIBE isolates (Billeter et al. 1994). In one MIBE case,50% of the uridine residues within the M gene weremutated to cytosines (Cattaneo et al. 1988). Thesemutations lead to protein truncations, elongations, ornonconservative amino acid substitutions (Schmid et al.1992). A general consensus at the time these reports werepublished was that these mutations, in some way, conferreda replicative advantage to the virus, perhaps making thisvirus more “neuroadapted” or neurovirulent. The advent ofreverse genetics, which allowed mutations to be selectivelyintroduced into wild-type strains, was a major step indiscerning what role these mutations played in viral growth.Interestingly, when a wild-type M protein was replacedwith a mutation-laden SSPE M protein, the virus was stillinfectious in neurons (Patterson et al. 2001), though thelack of a functional M protein did impair viral growthsomewhat. We have already alluded to a report thatsuggested that MV could apparently persist throughout lifewithout triggering overt disease (Katayama et al. 1995). Ifthis is true, sequencing of these viruses and comparingthem to isolates from MIBE or SSPE tissues might offerinsights into the functional consequences of biased hyper-mutations. Interestingly, a recent study using a greenfluorescent protein (GFP)-expressing, rodent-adapted MVshowed that, despite immunological resolution of MVwithout acute illness in adult immunocompetent mice, ahigh proportion of these otherwise healthy mice haddetectable MV-positive cells within the CNS weeks afterapparent clearance (Schubert et al. 2006), potentiallyparalleling the human autopsy studies described above.

Mouse studies

Because limited mechanistic insights can be gained fromhuman specimens, much of what we have learned about

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MV infection of the CNS, and resultant neuropathogenesishas come from animal models. As mice are not infectableby MV, mouse models of MV infection depend either uponthe use of rodent-adapted MV strains or artificial expressionof one of the human MV receptors, CD46 or CD150/SLAM. CD46 is now considered to be the principalreceptor for vaccine strains of MV, while CD150/SLAMis the primary receptor for wild-type strains, thoughexamples of overlapping receptor usage for both vaccineand wild-type viruses have been demonstrated.

Neuron-specific enolase (NSE)-CD46 mice were amongthe first transgenic mice to successfully express a humanMV receptor and be susceptible to infection (Rall et al.1997). In this model, transcriptional control of CD46 isgoverned by the NSE promoter, restricting expression andinfection to CNS neurons (Rall et al. 1997). A limitation ofthis system is that it does not recapitulate SSPE in humans,since no peripheral infection can occur, and susceptiblemice die during the acute phase of infection. However, ithas been useful to study MV–neuron interactions and tomeasure immune cell activation, recruitment, and functionwithin the CNS. Infection of NSE-CD46 mice by both anIC and IN route causes a neuron-restricted infection, thoughthe outcome of infection is predicated on the maturity andeffectiveness of the host immune response, as outlinedbelow. In addition to their utility as a model of pathogen-esis, the NSE-CD46 mice are a source of primary neuronsthat retain essential characteristics of this cell type(functional synapses, neuronal markers) and, further, arepermissive for MV infection. It is important to note,however, that, while these cells faithfully reproducecardinal aspects of neurons in vivo, the absence ofsupporting glial cells and the fact that these neurons arehippocampal in origin may not faithfully model MVinfection of other neuronal subtypes in the complex cellularenvironment of the brain.

CD46+ hippocampal neuron cultures can support MVreplication and spread, but the basics of the viral life cycleare fundamentally different from the standard textbookdescriptions. While infection of fibroblasts is characterizedby extensive release of extracellular virus, massive syncytiaformation resulting from cell-to-cell fusion, and concomi-tant target cell death, infection of neurons shares none ofthese features (Lawrence et al. 2000). Although the viruscan spread within neurons via trans-synaptic transmission(which is also true for polio, rabies, herpes simplex virus-1,pseudorabies virus, and other neurotropic viruses), noinfectious, free virus is released into the supernatant. Ofnote, free virus is not detectable in SSPE specimens either,though whether these observations are due to the samereplication restrictions is not known. Historically, theabsence of infectious virus and viral budding in humanSSPE brain specimens has been ascribed to the accumula-

tion of point mutations in the envelope-associated genes(Dubois-Dalcq et al. 1976; Rentier et al. 1978; Waters andBussell 1974). While these mutations may impact on viralrelease, they appear not to be essential for it to occur, sincevirus sequenced from CD46+ neurons is geneticallyidentical to input virus (Gechman and Rall, unpublishedobservations). This suggests that the neuronal microenvi-ronment alters the manner by which MV spreads, withoutnecessitating selection of adapted viral variants. Similarly,no neuronal syncytia are observed, perhaps due to aninability of the viral ribonucleoprotein to traffic to the cellsurface and acquire its envelope from the plasma mem-brane. Finally, and perhaps most strikingly, neuronsinfected with MV do not die of MV cytolysis: no differ-ences in cell death staining were observed between infectedand uninfected neurons, again resonating with humanstudies in which months to years of chronic infection canelapse with virtually no evident pathogenic impact.

These data implied that an alternative mode of viraltransmission was operative in neurons and that the CNSdisease observed in permissive mice could not be attributedto neuronal loss (Patterson et al. 2002). We have previouslysuggested (Rall 2003) that these key differences underscorethe concept that the viral particle and the infectious particleare distinct entities: the viral particle consists of the viralnucleic acids and the proteins that it encodes, whereas theinfectious particle consists of the viral proteins as well asthe necessary cellular proteins that the virus needs tocomplete its life cycle. Given that each cell type offers aunique set of “protein tools,” one can extrapolate that thevirus may encounter different sets of cellular proteins uponinfection that may alter (or prevent) completion of the lifecycle. For instance, Oglesbee and colleagues showed thateither transgenic expression of heat shock protein 72 inneurons or transient hyperthermia increased the levels ofMV RNA by as much as 2 orders of magnitude andcorrelated with increased neuropathogenesis (Carsillo et al.2006).

Studies on the trans-synaptic spread of MV revealedpotential roles for alternative MV receptors in neurons.While the CD46 receptor was required for initial viral entry,it was dispensable for neuron-to-neuron transmission(Lawrence et al. 2000), implying that the mechanism ofcell-to-cell spread—at least for neurons—was not the sameprocess as when a viral particle enters a non-neuronal targetcell. Moreover, a neurotransmitter receptor, neurokinin-1(NK-1), appears to be operative in trans-synaptic transmis-sion and may support the case for a receptor for MV-F,since the natural ligand of NK-1 receptor, substance P,bears homology to the active site of the fusogenic domainof F (Makhortova et al. 2007). Of note, Cosby andcolleagues showed that both wild-type and vaccine strainscan utilize alternative uptake mechanisms to gain access to

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both neurons and oligodendrocytes that do not expresseither CD46 or SLAM (Abdullah et al. 2009). Whileinfection in these murine cells occurred at a low level, thisstudy nevertheless reinforces the belief among many in themeasles community that more receptors await discovery.

Future pursuits

The unique life cycle that MV adopts upon infection ofneurons offers opportunities to learn more about neuronalbiology and the basis of chronic viral damage within thebrain. Some questions that remain include:

What specific neuronal proteins are associated withchanges in the MV life cycle? Movement of the MVRNP and envelope proteins to the neuronal synapsecould not occur by random cytoplasmic diffusion—there must be active transport from the site of synthesisin the perinuclear space to the nerve terminus. Thus,engagement of cellular motor proteins, includingdynein and kinesin that govern retrograde andanterograde transport, respectively, would likely berequired. Fairly little is known about how virusesengage these proteins, especially in a cell such as aneuron where the distance that needs to be traveledis so extensive. Recombinant MV-expressing markerproteins (e.g., GFP), coupled with new methods suchas slice cultures, will be crucial tools to help dissectthe replication and spread of MV in situ (Ludlow etal. 2008). Moreover, the use of other morbilliviruses,such as canine distemper virus (CDV), will beinformative: a GFP-expressing CDV was shown togain access to the CNS by both anterograde transportvia the olfactory bulb as well as hematogenous spreadthrough the choroid plexus and capillaries within thebrain (Rudd et al. 2006).What is the status of the virus during the long latencybetween initial exposure and disease? Autopsy studieswould suggest that the presence of MV in the CNS ismore common than the frequency of MV-triggeredCNS diseases would imply. Assuming this is true, itwould argue more in favor of the reactivation modelover the slow growth model. But what triggers viralresurgence? And how is the virus maintained in a latentstate? The issue of whether this is a ficommon eventwith a rare outcomefl, or a firare event with a commonoutcomefl remains to be defined.Is MV associated with chronic CNS diseases ofunknown etiology? Related to this prior point, and aswe noted earlier, MV has been associated with avariety of chronic diseases, though formal proof ofcausation of any disease with a neurotropic pathogenhas yet to be established. However, the notion that a

virus could trigger a CNS disease that looks quitedifferent from the acute disease is intriguing andcould possibly manifest as a result of direct viraldamage (to myelin-producing oligodendrocytes, forexample) or, indirectly, by induction of an autoreac-tive or overly aggressive immune response in theCNS. A lucid picture of how immune responsestypically function in the brain, which is discussed inthe next section, will be of key importance as thesestudies move forward.

Basic principle 2: neurons play a critical role in inductionof host immunity and noncytolytic clearance

When viral infections occur in CNS neurons, the immuneresponse faces a unique challenge. Since neurons arelargely a nonrenewable cell population, clearance bycytolytic mechanisms, such as through cytotoxic CD8+ Tcell interactions with infected cells, could cause irrevocableneuronal loss. In contrast, without an effective mechanismfor viral clearance, persistent infections of the brain couldbe easily established. Viral infections in the brain typicallyelicit a potent immune response that can include infiltrationof CD4+ and CD8+ T cells into the brain parenchyma,elevated levels of virus-specific antibodies in the CSF, andmicroglial activation. Yet, in many cases of CNS infection,viral clearance can occur in a noncytolytic manner withminimal damage to host CNS tissue (Binder and Griffin2001; Finke et al. 1995; Patterson et al. 2002; Rodriguez etal. 2003b). Our laboratory and others have identified acritical role for IFNγ, a pluripotent cytokine released byactivated T cells and natural killer cells, in this process(Finke et al. 1995; Patterson et al. 2002).

In the model of MV neuronal infection described above,adult NSE-CD46 mice require IFNγ in order to clear MVfrom neurons in the brain, as NSE-CD46 mice lackingIFNγ develop neurological disease, have widespread MVreplication in the brain, and succumb to infection (Pattersonet al. 2002). In recombinase-activating gene 1-deficientmice lacking B and T cells that express human CD46 underthe control of its endogenous promoter, adoptive transfer ofCD4+ T cells, could protect these immunodeficient micefrom MV-mediated neuropathology only if the CD4+ Tcells expressed IFNg (Tishon et al. 2006). Moreover, adultBalb/c mice control the spread of a rodent-adapted strain ofMV (CAM/RB) in the brain in an IFNγ-dependent manner.When IFNγ was depleted by antibody neutralization, Balb/c mice become susceptible to CAM/RB in the brain, andhelper T cells from these mice switched from a Th1 to aTh2 profile (Finke et al. 1995). Together, these studies point

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to a critical role for IFNγ in both directly controllingMV spread (and preventing subsequent MV-associatedneurological disease) as well as in effective induction ofthe host response.

To understand how IFNγ induces viral clearance inneurons, it is necessary to identify the downstreamsignaling pathways that are triggered upon IFNγ engage-ment. These pathways have been extensively characterizedin non-neuronal cells (reviewed in Stark et al. 1998) andthus will only be summarized here. Upon IFNγ binding andassembly of the receptor complex (consisting of a hetero-tetramer of IFNγR1 and IFNγR2 subunits), receptor-associated Janus kinases-1/2 are activated, resulting in thetyrosine phosphorylation of the cytoplasmic tail of theIFNγR1 subunits. Upon docking to the phosphorylated R1subunit, signal transducer and activator of transcription 1(STAT1) is phosphorylated on tyrosine 701, resulting in itshomodimerization. The phosphorylated STAT1 homodimertranslocates to the nucleus and binds to gamma-activatedsequence elements within IFNγ-responsive genes (ISGs) toinitiate transcription. These gene products establish the“antiviral state” in which the cell inhibits viral spread byupregulation of proteins that block viral gene transcriptionand protein translation, as well as those that cleave viralRNA. Notably, ISGs are induced in neurons and other CNScells during West Nile and LCMV infections, but ISGinduction (ISG-49, ISG-54, and ISG-56) only partiallydepends on STAT1 expression, suggesting that othersignaling pathways in CNS cells may contribute to ISGexpression and the antiviral state (Wacher et al. 2007).

Cell-specific responses to IFNγ have been noted in theregulation of STAT1 and in the activation of alternative,STAT1-independent signaling pathways (reviewed in vanBoxel-Dezaire and Stark 2007), suggesting that target cellsmay play a role in how they respond to IFNγ or to otherinflammatory cytokines. For example, many mitoticallyactive cells, such as fibroblasts, respond to IFNγ with rapidactivation of STAT1, followed by inactivation of STAT1 bysuppressor of cytokine signaling 1. In contrast, primaryhippocampal neurons respond to IFNγ with delayed andattenuated STAT1 phosphorylation and expression, thoughthe attenuated activation is sustained for days after theinitial IFNγ exposure (Rose et al. 2007). Since IFNγsignaling via STAT1 is often associated with antiprolifer-ative and proapoptotic effects, the attenuation in STAT1signaling in neurons may confer a survival advantage. Inaddition, many alternative signaling pathways have beenimplicated in IFNγ signaling, including activation of otherSTAT proteins and of other kinases such as protein kinase Cfamily members, PI 3-kinase, and p38-MAP kinase. Therole of alternative signaling pathways in IFNγ-mediatednoncytolytic clearance is unknown, but in light ofattenuated STAT1 activation by IFNγ seen in primary

hippocampal neurons, it is possible that neurons utilizeother signaling pathways in addition to STAT1 tomediate control of MV in neurons.

Even among neural cell types in the brain, IFNγdifferentially mediates viral clearance, depending upon theinfected cell type. For example, MHV infects astrocytes,microglia, and oligodendrocytes in the brain (Wang et al.1992); while perforin is sufficient to mediate viral clearancefrom astrocytes and microglia, oligodendrocytes dependupon IFNγ in order to clear MHV infection (Bergmann etal. 2006; Parra et al. 1999). In addition, retinal explantsfrom embryonic and neonatal mice strongly activateSTAT3, but not STAT1, upon IFNγ exposure (Zhang etal. 2005), confirming that neural cells can utilize alternativesignaling pathways in response to IFNγ.

IFNγ levels are rarely elevated in the CSF of adults orchildren with SSPE, suggesting that traditional mechanismsof viral clearance are either not activated or are suppressedin SSPE (Griffin et al. 1990; Ichiyama et al. 2006;Mistchenko et al. 2005). However, levels of IL-10, a Th2-related cytokine, are elevated in the CSF of SSPE patients,which could reflect an inappropriate skewing from a Th1response (IFNγ) to a Th2 response. T cells isolated fromthe peripheral blood of SSPE patients are largely unable toproduce IFNγ in response to MV infection in vitro, thoughIFNγ production in response to other viruses is notinhibited (Hara et al. 2000). Indeed, a subset of SSPEpatients from this study possessed T cells that were capableof producing IFNγ in response to MV similarly to non-SSPE controls. Those SSPE patients whose T cells werecapable of IFNγ production in response to MV maintainedreceptive function and progressed more slowly in compar-ison to SSPE patients with T cells that did not produceIFNγ in response to MV (Hara et al. 2000). These findingssuggest that, even in SSPE patients, where viral clearancefails in the brain, IFNγ may be beneficial in controllingMV spread and delaying disease progression.

Future pursuits

How does IFNγ mediate MV clearance/control fromdifferent cell types in the brain? While it is clear thatIFNγ is capable of controlling MV spread in the CNSand specifically in neurons, the signaling mechanismsthat ultimately lead to the control of MV are unknown.Understanding the downstream ISGs that are criticalfor MV control and whether there are cell type-specificdifferences between mitotically active cells and nondi-viding cells such as neurons, would offer great insightinto potential targets for treating SSPE.Why does MV control fail during SSPE? SSPE ischaracterized by widespread MV replication in thebrain, even though SSPE patients are not typically

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immunodeficient. How then does MV overcometraditional host defense mechanisms that are capableof limiting MV spread in other individuals?

Basic principle 3: developmental changes in the brainand immune response affect age-dependent pathogenesis

CNS complications with MV infection are overwhelminglyassociated with children, though this may reflect the youngage at which most people are typically infected. Given therelative paucity of adults with primary MV infections, it isdifficult to discern whether the development ofMV-associatedneurological complications is age-dependent. However, inanimal models of MV infection, young age correlates withboth severity of infection in the brain and pathologicaloutcome. For example, adult NSE-CD46 mice becomeinfected by MV in the brain following intracerebral orintranasal inoculation, but are able to clear the infectionwithin weeks without neurological damage, whereas neonatalNSE-CD46 mice demonstrate uncontrolled viral spread,develop seizures, and succumb to infection (Lawrence et al.1999). Of note, both adult and neonatal NSE-CD46 micerecruit approximately equivalent numbers of CD4+ andCD8+ T cells into the brain, and thus the age dependencedoes not appear to be due to an inability of neonates toinitiate an immune response. The basis of this differentialpathogenesis may be due to neuronal vulnerability: neonatalNSE-CD46mice show widespread apoptosis in the brain withastrogliosis and microgliosis throughout the parenchyma,while adult mice show little evidence of neuronal loss orinflammation (Lawrence et al. 1999; Manchester et al. 1999).

Other transgenic models expressing CD46 or SLAMhave shown similar age-dependent susceptibility to MVinfection in the brain. In transgenic YAC-CD46 neonatalmice, neurons are the initial target of MV, though low levelsof oligodendrocyte and microglia infection are seen later ininfection. As in the NSE-CD46 model, MV infection inneonates is ultimately associated with widespread MVreplication in neurons, T cell infiltration into the brain,apoptosis, and eventually death (Manchester et al. 1999;Oldstone et al. 1999). In another transgenic model, micewere established that ubiquitously expressed BC-Cyt1 or C-Cyt2 isoforms of CD46, the isoforms most abundant in thehuman brain. Ubiquitous expression of either isoformresulted in 100% mortality when neonates were infectedwith MV, again correlating with widespread infection andapoptosis (Evlashev et al. 2000). Interestingly, despiterobust CD46 expression in lymphocytes from these mice(more than in human lymphocytes), MV replication wasless in the murine lymphocytes than in the human

lymphocytes, suggesting that host factors other thanreceptor expression modulate the viral life cycle (Evlashevet al. 2001). Like CD46-expressing transgenic mice, micetransgenic for SLAM also demonstrate age-dependentsusceptibility; suckling mice infected intranasally withwild-type MV develop neurological disease and succumbto infection, while transgenic adults mount an immuneresponse to CNS infection and survive (Sellin et al. 2006).

While transgenic mouse models have been useful foranalyzing the immune components and neuropathogenesisof neonatal MV infections, none recapitulate the long lagbetween acute MV infection and CNS symptoms seen inSSPE. A model of chronic MV infection in the brain wasrecently established where young mice were infected with arecombinant MV virus expressing enhanced green flour-escent protein and the H protein from a rodent-adapted MVstrain (MV-GFP-CAMH) (Schubert et al. 2006) Replicationof GFP-expressing MV was then visualized in infectedneurons. Neonates succumbed to infection, but miceinoculated at 2 weeks of age survived and maintained apersistent infection in the brain out to 50 days post-infection, with viable GFP + neurons in the hippocampusand cortex. Though it is unclear how MV-GFP-CAMHpersists in immunocompetent mice when infected at ayoung age, this model provides an opportunity to dissectimmune components that may contribute to a chronic MVinfection in neurons.

Age is a major variable in the neuropathogenesis ofmany CNS infections. For many neurotropic viruses,cellular tropism becomes increasingly restricted as the brainmatures. Viruses such as LCMV, cytomegalovirus, andSemliki Forest virus readily infect areas of the brain that arerich in neural precursor cells and immature neurons, butbecome increasingly restricted to certain areas of the brainor to specific neuronal subtypes in the adult (Allsopp andFazakerley 2000; Bonthius et al. 2007; van den Pol et al.2002). In in vitro studies, MV readily infects undifferenti-ated NT2 cells, which are human teratocarcinoma cells thatcan be terminally differentiated into neurons with retinoicacid treatment (McQuaid et al. 1998). In contrast, differen-tiated NT2 neurons are initially refractory to MV infection,but can become infected by cell-to-cell contact withinfected neuroepithelial cells. In rodent models of MVinfection, MV spreads more aggressively in neonatalneurons than in adults, though whether this is due togreater tropism for immature neurons and/or to limitedimmune control is unknown. It is also unclear whether MVdemonstrates greater tropism for immature neural cells inhumans. Interestingly, in SSPE samples, MV antigens canbe found in many neural cell types, including neurons,oligodendrocytes, astrocytes, and brain endothelial cells,suggesting that MV is capable of infecting many types ofcells in the brain. If MV spreads from a limited presence in

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endothelial cells during acute infection to a more widelydisseminated infection in SSPE through trans-synapticspread or even if MV entry into the CNS occurs duringthe acute phase of infection in SSPE cases, remains to bedetermined.

In addition to changes in neural cells that may affect MVbiology in the brain, developmental changes in the immuneresponse may also contribute to age-dependent pathogene-sis. The efficacy of the neonatal innate and adaptiveimmune responses has classically been viewed as immatureor defective in comparison to adults when responding toviral infections. This is based upon observations that neonatalimmune cells differ quantitatively and qualitatively incomparison to adults. For example, neonatal T cells, whichare fewer in number in comparison to adults, were historicallyviewed as deficient due to their limited capacity to proliferateand produce IL-2 (Adkins et al. 2004). However, thishypothesis has been refined to include that the neonatalimmune response may be active, but skewed toward theproduction of “inappropriate” cytokines during viral infec-tions (Bot et al. 1998; Sarzotti et al. 1996). For example,human neonatal CD4+ T cells skew toward the production ofTh2 cytokines in response to environmental antigens,whereas adult CD4+ T cells from non-allergic individualsskew toward Th1 cytokines (Smith et al. 2008). In mice,neonatal CD4+ T cells show a Th1 deficiency, includingimpaired IFNγ production, even when adoptively transferredinto an adult host (Adkins et al. 2002). Collectively, theseresults suggest that neonatal adaptive immune responsemay struggle to control viruses whose clearance isdependent upon Th1 cytokines such as IFNγ. Given thecritical role that IFNγ plays in clearance from the CNS,as described in the previous section, neonatal defects inIFNγ production could impact on control of spread andclearance of MV.

Future pursuits

Why do infiltrating T cells fail to control MV infectionin the neonatal CNS? CD4+ and CD8+ T cells enterthe brain parenchyma during MV infection in adultsand neonates, but neonates cannot control MV spread.Recent work into neonatal T cells suggests that the Th2response dominates over the Th1 response during viralinfections, which could cause a deficiency in IFNγproduction and hence an inability to control MV, buthow this unfolds in the brain is unknown.What developmental changes in the brain and theimmune response contribute to the onset of SSPE? It islikely that MV enters the CNS during or soon afteracute infection, which typically occurs under 2 years ofage. Does the immune response interact with the

developing CNS in a fundamentally different way thanwith the mature CNS?

Concluding remarks

The point-of-view of this commentary is that critical basicobservations can be gleaned by studying rare diseases. Thethree main principles that were addressed here—the uniqueviral life cycle in neurons, the dissection of the non-cytopathic host response, and the potential basis of age-dependent pathogenesis—underscored progress that hasbeen made to date and highlighted key questions for futurestudy.

Our title referred to “blue moon” virology, meant toimply the study of diseases that arise infrequently. Indeed, ablue moon is, simply, an extra full moon that appearsduring the course of a year: 13 instead of the usual 12.Other than its relative infrequency, this extra full moon islike the others, illuminating in the same way as other fullmoons. We believe that the study of rare, virus-mediatedneurological diseases can afford insights into generalbiological and pathogenic processes, and in that way, canbe equally illuminating.

Acknowledgements We thank Kevin O'Regan, Christine Matullo,and Sarah Cavanaugh for advice on this manuscript and acknowledgesupport from the following sources: G.F.R. was supported by NIHgrants RO1-NS40500, RO1-NS060701, P30-CA006927, a PilotProject grant from Autism Speaks, Pennsylvania Department ofHealth Tobacco funds, and a gift from the F. M. Kirby Foundation.L.O’D. was supported by an NRSA Postdoctoral fellowship fromNINDS.

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