Immunology and Cell Biology

(2003)

81

, 207–216

Special Feature

Interaction of flaviviruses with cells of the vertebrate host and decoy of the immune response

N I C H O L A S J C K I N G

1

a n d A L I S O N M K E S S O N

2,3

Departments of

1

Pathology, and

2

Paediatrics and Child Health, The University of Sydney, Sydney, and

3

Department of Virology and Microbiology, The Children’s Hospital at Westmead, Westmead, New South Wales, Australia

Summary

Flaviviruses cause endemic and epidemic disease with significant morbidity and mortality throughoutthe world. In contrast to viruses that avoid the host immune response by down-regulating cell surface majorhistocompatibility complex expression, infection by members of the neurotropic Japanese encephalitis serogroupinduce virus-directed functional increases in expression of class I and II major histocompatibility complex andvarious adhesion molecules, resulting in increased susceptibility to both virus- and major histocompatibilitycomplex-specific cytotoxic T lymphocyte lysis. These changes are comodulated by T1 and T2 cytokines, as well asby cell cycle position and adherence status at infection. Infected skin dendritic (Langerhans) cells also showincreased costimulatory molecule expression and local interleukin-1

β

production causes accelerated migration ofLangerhans cells to local draining lymph nodes, where initiation of antiviral immune responses occur. The exactmechanism(s) of up-regulation is unclear, but changes are associated with NF-

κ

B activation and increased

MHC

and

ICAM-1

gene transcription, independently of interferon or other pro-inflammatory cytokines. We hypothesize thatthese viruses may decoy the adaptive immune system into generating low-affinity, self-reactive T cells which clearvirus poorly, as part of their survival strategy. This may enable viral growth and immune escape in cycling cells,which do not significantly up-regulate cell surface molecules. A possible side-effect of this might be immunopathol-ogy, caused by ‘autoimmune’ cross-reactive damage of uninfected high major histocompatibility complex andadhesion molecule-expressing cells, with consequent exacerbation of encephalitic disease. Results from a murinemodel of flavivirus encephalitis developed in this laboratory further suggest that interferon-

γ

plays a crucial role infatal immunopathology.

Key words

:

cell adhesion molecules, flavivirus, Langerhans cells, major histocompatibility complex, T-lymphocytes,West Nile virus.

Introduction

Haematophagous arthropod-borne viral infections of humansand animals have been of global concern for a considerableperiod of time and mosquito- and tick-borne flaviviral infec-tions in particular, continue to contribute significantly to thespectrum of disease caused by these. Yellow fever, caused bythe eponymous prototypic flavivirus, although well describedmore than two centuries before, came to public consciousnessduring the building of the Panama Canal between 1882 and1914, principally because of the delays it caused in construc-tion until about 1905 when the local mosquito population (atthat time, only just shown by Walter Reed as the link in itstransmission to humans) began to come under control.

1,2

Similarly, dengue virus caused a significant incidence of‘Breakbone fever’ during the American Civil War, althoughthe clinical disease was first described in North Americaalmost a century before that.

1

These single-stranded RNA viruses exhibit significantpleiotropism within the vertebrate host. They enter throughthe skin via the bite of an infected arthropod, proliferatinglocally and spreading to become generalized within a shortperiod of time, usually aided by a significant viraemia. Theviraemia enables transmission in turn to an uninfected arthro-pod vector, to maintain a pool of such infected vectors.Notwithstanding this ability to infect a wide range of cells, asubset of flaviviruses is in addition strongly neurotropic. Theprincipal members of this group of socio-economic impor-tance to humans include Japanese encephalitis, MurrayValley encephalitis, Kunjin, West Nile, Tick-borne encephali-tis, Louping ill and Saint Louis encephalitis viruses.

Each of these flaviviruses is endemic in different regionsof the world. However, significant epidemics occur outside ofthese areas. This is illustrated by the recent spread of WestNile virus (WNV) throughout the United States, beginningwith an epidemic in New York in August 1999.

3–5

Formerly,WNV was endemic in mostly developing countries, princi-pally in regions of India, Africa and Europe, with periodicoutbreaks in adjacent or nearby regions. One of the Japaneseencephalitis serogroup, epidemic WNV produces an encepha-litic syndrome which is fatal in approximately 10% ofclinically apparent cases, while many survivors may havepermanent neurological sequelae. With global warming

Correspondence: Dr Nicholas King, Pathogenesis Group, Labora-tories of Viral Immunology, Department of Pathology, D06, Univer-sity of Sydney, Sydney, NSW 2006, Australia.Email: nickk@pathology.usyd.edu.au

Received 25 March 2003; accepted 25 March 2003.

208

NJC King and AM Kesson

extending the environment permissive for arboviral vectorsand the increase in world travel by humans, a resurgence inarboviral disease has occurred in the last two decades. Withthe likely continuation of this trend, flaviviral disease willbecome increasingly important.

6–8

Vaccination against several neurotropic flaviviruses hasshown promise, most recently with chimeric vaccines.

9,10

However, treatment of non-immune virus-infected individu-als with clinical disease remains a problem. In these cases,understanding the mechanisms of pathogenesis of disease isthe key to informed and effective clinical treatment andsupport.

The cellular response to flavivirus infection

The arthropod-borne flaviviruses maintain a high degree ofgenomic stability, presumably due to the enzymatic and othermolecular requirements for replication in both homoiothermicvertebrates and poikilothermic arthropods. The use of parallelhosts, i.e., multiple species with varying reproductive rates,and the more-or-less carrier status of some species, undoubt-edly contribute to meeting the requirement for transmissionand replication of these viruses through a continuing pool ofnon-immune hosts. However, the lack of selective geneticmutation, recombination and/or drift, clearly associated withsurvival success in many other viruses, suggests these flavi-viruses have evolved a different set of strategies for inter-action with the immune response.

Interactions with the host immune response that result inan increase in the time available for virus replication beforehost immunity (or death) occurs are likely to maximize thehost output of progeny virus. Interactions occur with theinnate and/or the adaptive immune systems by a variety ofvirus-directed soluble or membrane-bound decoy moleculesthat directly or indirectly inhibit the generation or prosecutionof the antiviral immune response.

11–15

In the adaptive immuneresponse, viral peptides are specifically recognized by T cellsin the context of cell surface major histocompatibilitycomplex class I or II (MHC-I/II) molecules via the T cellreceptor (TcR). This is further refined by CD8 or CD4molecules on the T lymphocyte, which specify MHC-I or IIrecognition, respectively, on infected target cells. The affinityof specific MHC/virus peptide recognition is variable. How-ever, the avidity of this interaction is potentiated by CD8 andCD4, as well as an array of adhesion molecules, particularlyintercellular adhesion molecule-1 (ICAM-1, CD54) and vas-cular adhesion molecule-1 (VCAM-1, CD106),

16,17

recog-nized by the integrin receptors, LFA-1 (CD18/CD11a) andVLA-4 (CD49d/CD29), respectively, also found on T lym-phocytes. Major histocompatibility complex and ICAM-1/VCAM-1 are members of the Immunoglobulin Superfamilyand are constitutive and/or inducible variously, on mostsomatic cells by a wide range of cytokines. Thus IFN-

γ

(type2 IFN) and a range of interleukins produced by T cells, aswell as other more directly antiviral cytokines such as IFN-

α

and

β

(both type 1 IFN) and TNF produced by virus-infectedcells, strongly up-regulate these molecules. These cytokinesalso normally feed back into the developing immune response.

18–20

Increases in cell surface concentration of MHC and adhesionmolecules, individually or together on infected cells, signifi-cantly increase the probability of their adhesive interaction

with T lymphocytes. Such cells readily become targets ofimmune attention, efficiently recognized by T lymphocytesexpressing the cognate TcR.

21–25

In this way, increased MHC-Iexpression on virus-infected cells results in increased lysis byvirus-specific CTL

26–29

which are critically important in thecontrol of virus by the host. Interestingly, cytokines producedby activated immune lymphocytes, such as IFN-

γ

, are gener-ally much more potent at up-regulating cell surface immunerecognition molecules than cytokines produced much earlierby infected cells. This suggests a temporal and mechanisticrequirement for a staged up-regulation of these moleculesduring the generation of an effective immune response result-ing in virus clearance.

The recognition of MHC/virus peptide is the pivotal stepand a

sine qua-non

in generating an adaptive antiviralimmune response. Not surprisingly, molecular mimicry of‘self’ by virus peptide/MHC combinations measurablyreduces the array of antiviral defences.

30

Predictably also,down-regulation of MHC expression at the cell surface byviruses is one of the most potent ways of reducing immuneresponses.

31

Reduced or absent MHC effectively produces‘immunological silence’. This occurs, for example, withvaccinia

32

and herpesviruses,

31,33

as well as adenoviruses.

34,35

Interactions with the host immune system often require thetranscription of specific molecules encoded by the infectingvirus. Such genes are usually analogous to crucial hostimmune response-modulating genes and have presumablybeen acquired over years of monogamous coevolution withspecific hosts. However, the broad species specificity andpleiotropism of flaviviruses may have limited the acquisitionof extraneous genes, with the result that the compact flavi-virus genome is concise, containing little else but the genesevidently necessary for successful replication and trans-mission in the requisite hosts.

36

Flavivirus infection increases MHC and adhesion molecule expression

We have been investigating the interaction of flaviviruseswith vertebrate host cells for several years now. In contrast tothe effects of many viruses, a striking result of infection of allspecies and all cells tested in our laboratory is the significantup-regulation of cell surface expression of MHC-I, dependingon the type of cell infected and the length of time ofinfection.

28,37–44

We have found this response occurs withWest Nile virus, Murray Valley encephalitis, Kunjin andJapanese encephalitis, but it also occurs with yellow fever anddengue.

45

While some of this up-regulation is clearly attributable toIFN-

β

activity, it is clear that a significant part of this is virus-directed. Initial experiments using neutralizing polyclonalantibody to inactivate IFN-

α

/

β

only partially blocked the up-regulation of MHC-I caused by WNV infection.

40

Cells inwhich IFN activity is developmentally absent, also up-regulateMHC-I in response to WNV infection. Thus, WNV-infectedtrophectoderm of the preimplantation blastocyst increases cellsurface MHC-I expression, despite being both unresponsiveto IFN-

α

/

β

and IFN-

γ

and unable to produce thesecytokines.

41

Moreover, MHC-I is still up-regulated in WNV-infected fibroblasts isolated from type 1 IFN receptor knock-out mice. These cells produce type 1 IFN but cannot use it and

Flavivirus decoys the immune response

209

are consequently much more susceptible to flavivirus infec-tion compared to wild type control cells (Cheng Y

et al.

unpubl. obss).

In vitro

, WNV-induced MHC increases are significantlygreater in mouse embryo fibroblasts (MEF) infected in G

0

,than in MEF infected during other phases of the cell cycle.Interestingly, the contribution to MHC-I up-regulation byIFN-

β

is greater in WNV-infected cycling MEF than in thosein G

0

. Most of the MHC-I increase associated with WNVinfection in cycling cells can be abrogated by neutralizingantibody to type 1 IFN. Cycling cells are more susceptible toWNV infection than cells in G

0

and a greater percentage ofcycling MEF become infected, compared to cells in G

0

.Nevertheless, on a cell-for-cell basis, infected cells in G

0

produce a similar amount of IFN-

β

to infected cycling cells.Moreover, uninfected cells in G

0

are in themselves lesssusceptible to the MHC-I-increasing effects of IFN-

β

thancycling cells. These differences are reflected functionally inan approximately 10-fold increase in the susceptibility ofWNV-infected G

0

cells to CTL lysis by virus-specific andallo-specific CTL.

27

As might be expected, cycling cells areconsiderably more productive of WNV than quiescent cells.

29

Major histocompatibility complex class II is also up-regulated on several cell types in response to WNV infection.This may be increased on cells already expressing this mole-cule constitutively or induced

de novo

in cells with nodetectable constitutive expression. Up-regulation of MHC-IIon human myoblasts

38

and endothelial cells,

44

rat schwanncells

37

and murine astrocytes,

46

macrophages

43

and Langer-hans cells,

39

suggest that this response is broadly based. Onthe other hand, MHC-II expression is not induced by WNVon fibroblasts from human (despite increased MHC-II expres-sion in response to IFN-

γ

in these cells) or mouse cells (whichare not responsive to IFN-

γ

). This indicates that this responseis limited to particular cell types, presumably those with themolecular signalling pathways required by the virus, asopposed to those required by IFN-

γ

. West Nile virus-mediatedMHC-II induction is independent of MHC-II-inducingcytokines and, similar to WNV-induced MHC-I, is functionalat the cellular level.

46

West Nile virus also induces significant up-regulation ofseveral non-polymorphic cellular adhesion molecules. Immu-noglobulin superfamily, ICAM-1 (CD54) and VCAM-1(CD106), are both up-regulated 2–3-fold within 2 h andmaximally (4–6-fold) by 4 h of WNV infection both

in vitro

44

and

in vivo

39

(Shrestha B and King NJC unpubl. obss). In theselectin family, E-selectin (CD62E) is also up-regulated withvery short response times (30 min) in endothelial cells,achieving maximal up-regulation at 10-fold the baselineexpression within 2 h of WNV infection. On the other hand,P-selectin (CD62P), normally stored for immediate release inthe Weibel-Palade bodies in endothelial cells, is evidently notaffected in the short term by WNV and is up-regulated lessthan 2-fold in 4 h (Fig. 1). Importantly also, expression ofCD44, a homing cellular adhesion molecule, as well as therelevant receptors for the above molecules are not up-regulatedby WNV infection (King NJC, unpubl. obss). Thus, this up-regulation is not a widely ranging non-specific up-regulationof cellular proteins.

29,47

In the case of ICAM-1, WNV-induced expression, likeMHC-I, is dependent on cell cycle status. This response is

almost absolute; human fibroblasts infected in G

0

increaseICAM-1 maximally by 5–6-fold, while the increase in ICAM-1expression in cycling cells is close to zero. Moreover, thisresponse to WNV in G

0

cells is effectively quantal. Intercellu-lar adhesion molecule-1 increases only occur with a multi-plicity of infection of five or more plaque forming units percell and this dose produces approximately 60% of the finalmaximal increase possible with higher virus concentrations.Major histocompatibility complex class-I up-regulation iscontinuous, with a predictable virus dose–response curve,undoubtedly achieved by the smoothing effect of type 1 IFN.Intercellular adhesion molecule-1 is barely responsive to type1 IFN.

42

It follows that this cytokine plays little part in ICAM-1up-regulation by WNV. This is consistent with our findingthat WNV directly mediates most of the MHC-I up-regulationin G

0

cells, while IFN-

β

produces most of the MHC-I up-regulation in cycling cells.

Like MHC-II, up-regulation of these adhesion moleculesseems to be mediated by WNV alone, since neutralizingantibodies to various candidate cytokines had no effect on thisincrease.

44

However, it is of interest to note that the increasedexpression of these molecules is further modulated by expo-sure of WNV-infected cells to a range of immuno-activecytokines. Modulation generally occurs according to the pro-or anti-inflammatory influence of the cytokine. Thus T1cytokines, such as TNF, may further enhance cell surfacemolecule expression induced by WNV infection, while T2cytokines, such as IL-4 may inhibit it.

44

Similar to MHC-I andII, up-regulation of adhesion molecules is functional. WestNile virus-infected cells show significant increases in infectedcell-activated leukocyte adhesion over uninfected cells (KingNJC, unpubl. obss).

All in all, our findings strongly suggest that up-regulationof these immune recognition molecules is a virus-directedeffect which drives transcription of the relevant genes (seebelow) and leads to subsequently increased cell surfaceexpression, rather than a host-mediated antiviral response.The implications of variation in the amplitude and kinetics ofincreased adhesion molecule expression and their subsequent

Figure 1

Composite graph showing the kinetics of up-regulationof MHC-I (

�

), MHC-II (

�

), ICAM-1 ( ), VCAM-1 (

�

),E-selectin (

�

) and P-Selectin (

�

) on human umbilical veinendothelial cells in response to infection by West Nile virus(WNV). The abscissa shows a broken scale of hours. The ordinaterepresents the fold increase in level of expression of each mole-cule. The broken line in each graph represents the change in timescale.

210

NJC King and AM Kesson

modulation by immune-active soluble factors may be playedout

in vivo

during viraemic episodes which result in thespread of the virus to the rest of the body. It is possible thatvirus carried in monocytes or dendritic cells (DC) may spreadvirus in their passage through the blood stream and lymphat-ics via adhesive interactions (Fig. 2). Adhesion status, closelyassociated with activation and differentiation in both thesecell types, influences the cellular response to WNV. Forexample, no up-regulation of MHC-I and II and ICAM-1occurs in WNV-infected macrophages kept under nonadhe-sive conditions

in vitro

. Indeed, MHC-II expression is down-regulated. However, expression of these molecules may be‘superinduced’ when adhesive conditions supervene.

43

Sincethere is no difference in susceptibility to WNV infectionunder adherent and nonadherent conditions, it is possible thatvirus activity is kept ‘on hold’ in nonadherent cells untiladhesive interactions between these cells and mesenchymalcells can legitimately occur

in vivo

. It is also likely thatendothelial cells infected during viraemia would increasetheir adhesive capacity for particular leukocyte types atvarious times and could enhance the spread of virus else-where via these migratory cells. This may be further exacer-bated by the release of a range of soluble factors frominfected endothelial cells and/or leukocytes, including chemo-kines, cytokines and nitric oxide, which may potentiate suchinteractions variously, by attracting particular leukocytesubsets in the first place, by modulating cell surface expres-sion of adhesion molecules and by altering adhesive inter-actions.

43,44,48

We are currently investigating these possibilities.

Mechanism of flavivirus-mediated MHC and adhesion molecule up-regulation

How WNV causes the direct and dramatic increases in theexpression of MHC and adhesion molecules, is of obvious

interest. Others have suggested that WNV-induced MHC-Ioccurs as a result of an increase in peptide supply to theendoplasmic reticulum.

49

While evidence supports this as onepossible mechanism for MHC-I up-regulation, the consistentinduction by WNV of such a variety of cell surface immunemolecules argues for a mechanism common to all thesemolecules.

We have shown that WNV induces increased

MHC-1

47

and

ICAM-1

29

gene transcription. Significant increases inMHC-I and ICAM-1 mRNA are unaffected by the presenceof neutralizing polyclonal anti-interferon-

α

/

β

antibody duringinfection, indicating that this is not a type 1 IFN-drivenphenomenon.

29

Furthermore, up-regulation of MHC-I cellsurface expression does not occur when transcription isblocked with actinomycin D in WNV-infected cells.

40

How-ever, the increase in cell surface expression of molecules suchas E-selectin and the early increases seen in ICAM-1 arelikely to be too rapid to be the result of novel gene transcrip-tion in WNV-infected cells. Thus whether, in addition,mRNA for these molecules is in some way disinhibited orstabilized by WNV infection to prolong its half-life, initiallyup-regulating and subsequently augmenting increased cellsurface expression, is a matter of speculation.

NF-

κκκκ

B is activated by WNV infection

Nuclear factor-kappa B (NF-

κ

B) is an inducible eukaryotictranscription factor which belongs to the highly conserved

rel

- related family. Nuclear factor-kappa B is a homo- orhetero-dimeric complex made up variously, of one of anumber of permutational possibilities from the subunits, NF-

κ

B1 (p50), NF-

κ

B2 (p52), relA (p65), Rel B and c-Rel. It isretained in an inactive state in the cytoplasm through theinteraction with inhibitor-

κ

B (I

κ

-B). Phosphorylation of I

κ

-Bactivates NF-

κ

B which immediately translocates to the cell

Figure 2

Simplified depiction of the putative process of flavivirus infection from entry of the virus via the skin to clearance and/orimmunopathology.

Flavivirus decoys the immune response

211

nucleus. Here, different NF-

κ

B complexes have differentaffinities for a range of homologous decanucleotidesequences (5

′

-GGGRHTYYCC-3

′

) in various gene promoterregions, thus controlling the transcriptional activity of a widerange of genes. These include MHC-I, MHC-II, ICAM-1 andVCAM-1, as well as a great many proteins induced duringinflammatory and immune responses (reviewed by Baldwin).

50

Upon infection, WNV induces NF-

κ

B activation andnuclear translocation. This activated complex comprises aheterodimer of p65 and p50 and can readily be demonstratedby electro-mobility shift assays (EMSA)

47

and confocalmicroscopy of immunohistochemically labelled cell mono-layers (Cheng Y, unpubl. obss). If phosphorylation of I-

κ

B isinhibited in WNV-infected MEF with the protein kinase Cinhibitor, H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine],or salicylates, the block to NF-

κ

B activation results in ademonstrable reduction in NF-

κ

B nuclear translocation andabrogates the increase in cell surface expression of MHC-I

47

and ICAM-1 (Kesson A, unpubl. obss).These effects are not due to the classical up-regulation of

type 1 IFN seen in our experiments, since this cytokine doesnot induce significant levels NF-

κ

B in these cells.

47

Nor is itdue to the induction of TNF, which is a potent inducer ofNF

κ

B and which we have recently shown is induced at bothmRNA and protein level in WNV-infected MEF. Both thekinetics of induction and the subunits induced by TNF aredifferent from those induced by WNV. Furthermore, WNV-induced NF-

κ

B activation and MHC-I up-regulation occurs inthe absence of both these cytokines in cells from interferontype 1 receptor and TNF gene knock out mice. This demon-strates that WNV-induced NF-kB activation and subsequentup-regulation of MHC-I is independent of the secretion ofthese cytokines (Cheng Y

et al.

unpubl. obss).Whether the activation of NF-

κ

B is useful or required forproductive WNV infection is not clear. Current experimentsaddressing these issues are in progress.

West Nile virus infection

in vivo

The functional up-regulation of cell surface immune recogni-tion molecules suggests that infected cells would have a highimmunological profile in vivo. We have therefore, developedtwo murine models to investigate this issue in detail. The firstis a model of skin infection that attempts to approximate theroute of normal acquisition of virus, the second is a patho-genesis model of encephalitis.

West Nile virus causes increased migration of Langerhans cells

Langerhans cells (LC) occur at a frequency of 1–3% of cellsin the epidermis. They are intercalated evenly between thekeratinocytes throughout the epidermis, appearing to be indirect contact with one another via an extensive network ofcytoplasmic dendrites. Work with contact sensitisers showsthat LC up-regulate MHC-I, II, ICAM-1 and costimulatorymolecules, CD80 and CD86, and reduce E-cadherin expres-sion, prior to migrating to the local lymph node to present thesensitizing antigen.51,52 A model of WNV infection of ear skinis being used in our laboratory to investigate the initiation of

anti-WNV responses via LC. Using this model, we haveshown that LC respond to WNV infection, either in vivo orin vitro by up-regulating MHC-I, II, ICAM-1 and CD80.39

This alteration in cell surface phenotype, denoting a changefrom an antigen-processing to antigen presenting function, isaccompanied by a significant increase in migration of LCfrom the skin to the local draining lymph nodes, as measuredboth by the reduction in LC in the skin and an increase inFITC-labelled LC in the matched lymph node. This migrationis faster than that observed after skin infection with SemlikiForest virus, an alphavirus (a single plus-sense RNA virusalso transmitted by mosquitoes). Moreover, WNV-inducedchanges occur some 8 h before the influx of leukocytes to thedermis below the local area of infected skin. Of considerableinterest is the finding that UV-irradiated virus of either typefailed to cause any of these changes. Thus, it is clear thatdespite the ability of LC to respond to non-replicating anti-gens such as contact sensitisers, these cells can distinguishbetween replicating and non-replicating virus.53 Furtherinvestigation subsequently demonstrated that LC are inducedto migrate after WNV infection in response to IL-1β,54 despitethe requirement for both IL-1β and TNF in contact sensitisersystems.55 Our current investigations are continuing to definethe role of the local environment in the initiation of LCmigration after virus infection, as well as that of LC in theinitiation of WNV-specific antiviral immune responses.

The pathogenesis of encephalitis

In addition to the commonality of NF-κB driving up-regulated expression of most of the molecules up-regulatedby WNV, most of these molecules are also significantly up-regulated by IFN-γ, produced by activated immune lympho-cytes recognizing cognate antigen. As part of the hostresponse to virus infection, this cytokine is frequently crucialto viral clearance and resolution of virus infection.56,57 There-fore, we have developed a model to investigate the require-ment of this cytokine in WNV encephalitis. In adult C57BL/6IFN-γ gene knockout (B6.IFN-γ–/–) and matching congenicC57BL/6 wild type (B6.WT) controls, intraperitoneal admin-istration of WNV producing mortality in the mid-range inwild type mice was reduced to less than one third in theB6.IFN-γ–/– group. Kinetics of infection was similar in bothgroups: both showed detectable WNV in the brainstem on day6, spreading from caudal to rostral, both had similar numbersof infected neurones peaking on days 7 and 8 postinfectionby immunohistochemistry. However, twice the number ofB6.IFN-γ–/– showed detectable neuronal WNV infection betweenday 6 and 11, compared to B6.WT mice. No other cell type inthe brain was infected in either strain.

An infiltration of leukocytes was also present in the brainparenchyma of mice with WNV encephalitis. Neutrophils andmononuclear cells comprised the majority of these and by day7 post infection, this infiltrate was 4-fold greater in B6.WTthan B6.IFN-γ–/– mice. This differential in infiltrating cellpopulations occurred despite significantly greater vascularendothelial ICAM-1 and VCAM-1 expression in the brains ofB6.IFN-γ–/– mice compared to B6.WT mice.

In both strains the numbers of activated microglia in brainsections were increased, but were 3-fold higher in B6.IFN-γ–/–

than in B6.WT mice at the peak of activation. Activation,

212 NJC King and AM Kesson

determined by phenotypic and immunohistochemical criteria,was detectable by day 7 onwards, with obvious noduleformation around infected neurones. Neuronal death was rare,however, and was detectable in no more than 0.1% of infectedneurones by terminal deoxynucleotidyl-transferase-mediateddUTP-biotin nick end labelling (TUNEL) in either group.Finally, rechallenge in survivors from both strains with WNVat doses 50-fold higher than initially used, resulted in noclinical or histological evidence of disease (Shrestha B et al.unpubl. obss).

West Nile virus (Sarafend) encephalitis in the B6.WTmodel thus exhibits the histopathological and clinical featuresof human disease58 and as such, is relevant to the study ofWNV in the murine model. The important findings from theB6.IFN-γ–/– mouse are:1. an increase in survival, without any decrease in viral load2. a reduction in leukocyte infiltration into the brain,

although critical leukocyte adhesion molecules weremarkedly up-regulated

3. significantly increased microglial activation and micro-glial nodule formation and

4. effective immunity with virus clearance, all occurring inthe genetic absence of IFN-γ.These results thus indicate that IFN-γ, and/or its subse-

quently induced downstream factors, are crucial for effica-cious leukocyte recruitment into the brain parenchyma inWNV encephalitis. Massively up-regulated neurovascularendothelial adhesion molecule expression in the absence ofIFN-γ, does not recruit the usual complement of leukocytesinto the brain. The activation of microglia, their migration andformation of nodules around infected neurones are also inde-pendent of IFN-γ. Although it is possible that the activatingstimulus comes from infiltrating leukocytes, the more intenseactivation of microglia in the face of a lesser leukocyteinfiltration in the B6.IFN-γ–/– mouse makes this unlikely.Moreover, inoculation of either strain with WNV via theintranasal route in both mouse strains results in activation ofmicroglia 48 h before leukocyte infiltration. This suggeststhat infection of neurones and/or the immediately accompany-ing downstream events are responsible for this stimulus ratherthan infiltrating leukocytes responding to infection (ShresthaB et al. unpubl. obss).

Our findings highlight the overlap in function between thevariety of soluble mediators produced as a response to virusinfection in normal circumstances. In WNV encephalitis inthe absence of IFN-γ, however, leukocyte recruitment issignificantly reduced. A murine model of Yellow feverencephalitis in the same mouse strains showed similar results,although the differences were not as big.59 For leukocytes tobe maximally recruited in these models it may thereforerequire the presence of chemotactic signals induced directlyor indirectly by IFN-γ.60,61

These results also show that the generation of both anefficient sterilizing adaptive immune response and effectiveimmunological memory can still occur in the absence of IFN-γ.Whether the immune response in the absence of IFN-γ isprincipally comprised of elements more consistent with a T2immune response rather than T1 type in WNV encephalitisremains to be elucidated, and is being further investigated inthis laboratory.

Most importantly, however, these results suggest that IFN-γmay either directly or indirectly cause fatal immunopathologyduring WNV encephalitis.

Virus-directed, increased cell surface molecule expression, increased immunological profile and immunopathology – a paradox?

Evidence to date leads to the evolutionarily untenable butinescapable conclusion that WNV enhances its own eradica-tion by flagging itself to the adaptive immune system inmammals. The immune response is initiated via an acceler-ated Langerhans cell migration to the draining lymph nodeafter skin infection.53,54 Major histocompatibility complex andvarious adhesion molecules, including costimulatory mole-cules, are up-regulated or induced de novo on a variety ofcells which either induce the antiviral immune response or aretargets for it.37–44,46 Virus-specific CTL generated in vivorecognize and kill WNV-infected cells more efficiently thelonger they have been infected, reflecting both this increasingfunctional expression and the fact that virus-specific peptideis clearly not limiting in CTL recognition.27–29,47 Finally, theimmune response thus generated is associated with a signifi-cant amount of immunopathology, which may lead to death ofthe host and which may be the result of this increased immunevisibility.62

It is possible that these effects only occur in so-called‘dead-end hosts’, i.e., vertebrate hosts in which viraemia isnot thought to attain sufficiently high levels to be transmittedto the arthropod vector. Work is only beginning on thedetailed comparative immune response to these viruses in thebird populations constituting the reservoir for the usual cycleof vertebrate-arthropod-vertebrate transmission.63 However, itis difficult to believe that such a fundamental and broad-basedvirus-directed effect in mammalian cells will not be replicatedin avian cells. Moreover, it is tempting to speculate theincrease in bird mortality in the USA associated with thespread of WNV throughout the USA is a result of interactionof WNV with a genetically unfamiliar host population thathas not yet undergone evolutionary selection to minimizethese immunopathological effects.

Another possible explanation for this apparently increasedimmune profile, for which there is in vitro evidence, is thatup-regulated MHC inhibits NK cell lysis.64 Unpublished workalluded to in this edition reports that lack of NK cell activityhas little or no effect on the outcome of infection,65 implyingthat the virus effectively abrogates the NK cell responsein vivo in normal mice. However, while this strategy wouldundoubtedly be useful for the virus in the initial stages ofvirus infection as part of the innate response, it seems unlikelythat there are not other downstream and/or later events thatalso result in the avoidance of the immune response, eithertied to abrogation of NK cell activity or acting separately.Indeed, the evident involvement of the adaptive immuneresponse in the pathogenesis of disease in flavivirus infectionsuggests that part of the answer lies with the generation of thespecific antiviral immune response. Therefore, we proposeanother explanation for the high profile of flaviviruses pre-sented to the adaptive immune response.

Flavivirus decoys the immune response 213

Flavivirus evasion of the immune response – an hypothesis

In the generation of an antiviral response, there is a widerange of affinities in polyclonal virus-specific CTL popula-tions, including those for allo-MHC and heterologous virusantigens66,67 although not completely unrelated antigens.68

Increases in MHC-I that occur on WNV-infected cells clearlyenhance the avidity of the CTL-target interaction.26,27,29 Withthe additional up-regulation of costimulatory molecules, suchas those that are induced by WNV in Langerhans cells inchanging from an antigen processing to antigen presentingphenotype and migrating from the skin to the draining lymphnode,39,53,54 the increase in avidity would be expected torecruit a wider range of low affinity CTL, which wouldotherwise be below the recognition threshold with normalexpression of such cell surface molecules.17,22,24 Up-regulatedexpression in WNV infection could therefore result in thegeneration of a larger range of CTL clones than is usual forantiviral responses, with accordingly varying affinities forMHC-I + virus peptide. Predictively, such populations wouldstatistically include low affinity, self-reactive CTL clones.Since MHC-I-specific CTL can recognize MHC-I withoutpeptide specificity69 with the increased avidity between Tcells and high MHC/ICAM-expressing cells, such cloneswould be able to kill both infected and uninfected target cellsthat expressed high levels of MHC-I. Initially, MHC wouldbe increased only on infected cells. However, the secretion oftype 1 IFN by infected cells would increase the expression ofMHC-I on all cells in the vicinity and this up-regulationwould be potentiated by the subsequent secretion of IFN-γ byactivated T cells recognizing cognate antigen with high avidity.

If low affinity, self-reactive clones are generated, wewould expect to see significant cross-reactive lysis of un-infected targets that express high MHC-I concentrations(reviewed by Murray and McMichael).70 Indeed, our experi-ments show that ‘WNV-specific’ CTL lyse IFN-γ-treated,uninfected MEF expressing high MHC-I concentrations moreefficiently than uninfected, untreated MEF.29

Furthermore, high affinity CTL require little or no stabili-zation of the interaction between the MHC-I and the TcRfrom additional accessory molecules, such as CD8, to enableCTL-target cell signal transduction. On the other hand, stabi-lization may be required when CTL affinity is low.22,24 It islikely therefore, that the CD8-MHC-I interactions are impor-tant during recognition of WNV-infected cells by low affinityWNV-specific CTL.71 Moreover, non-specific accessory mol-ecules like ICAM-1 on the target cell, interacting with itsreceptor, LFA-1 on the T cell, can also add to the stability ofT cell-target interactions.16,17 As avidity can be increased viaboth specific, i.e. MHC-I-associated, and non-specific, e.g.CD8-MHC-I, ICAM-1–LFA-1 interactions, comodulation ofMHC and ICAM-1 would be expected to further enhance thebroad host cellular immune response against WNV, thusincreasing the probability of generating low-affinity self-reactive CTL.

If we add to this, the possibility that a variable range ofquasispecies may be generated during infection, this wouldresult in an even wider range of T cell clones being generated,with the commensurate range of variation in their TcRaffinities.36,72–74

Low-affinity self-reactive recognition by CTL wouldoccur to a greater extent in interaction with cells infected byWNV in G0, which express very high levels of MHC-I, whileWNV-infected cells in other phases of the cycle, expressinglower levels of MHC-I, would be less easily recognized bylow affinity CTL. This would be further amplified by thedifferential effects of type 1 and 2 IFN on the target cells indifferent phases of the cell cycle. Thus, although IFN-β has amore pronounced effect than WNV on MHC-I expression ininfected cells in G1 (with relatively little effect on cells in G0),its effect on MHC-I expression in G1 is not as great as that ofWNV on cells in G0. Moreover, IFN-γ has a significantlygreater effect on cells in G0 than cells in G1.

27 Thus, thecombined effects of WNV and cytokines clearly supporthigher expression of MHC on G0 cells and lower expressionon G1 cells.

We therefore, speculate that since most cells in vivo are inG0, a minority of WNV-infected cells in other phases of thecycle may escape detection by WNV-induced low-affinity Tcell clones of the host immune system for a sufficient periodof time to allow for replication of virus and transmission to aninvertebrate host. Infected G0 cells therefore, may effectivelydecoy the immune system to recognize and respond to themstatistically in preference to infected cells in G1. From anevolutionary point of view, this would predict that greaterproduction of virus might occur in G1 cells to subserve therequirement for successful replication and subsequent trans-mission. Indeed, we have found WNV replicates in vitro up toa 15-fold higher titre in cycling cells than in cells in G0. Thus,non-confluent WNV-infected MEF produce significantlygreater amounts of infectious WNV on a cell-for-cell basisthan their confluent G0 counterparts.29 In vivo, such discrepan-cies in WNV replication in quiescent versus cycling cellswould further enhance the possibility of virus transmissionwhile cycling cells maintained a relatively low immunologi-cal profile compared with quiescent cells. If the associatedillness were incapacitating, this would in addition promoteunimpeded access by the arthropod vector to the host duringthis period. Experimentally, high affinity virus-specific Tcells show far better virus clearance in vivo, while, even inhigh numbers, low affinity antiviral T cells clear virus com-paratively poorly.75 Obviously with time, high affinity T cellclones would proliferate sufficiently to clear virus from bothhigh and low MHC-expressing infected cells, resolving theinfection and leaving the host immune (and alive).

As implied above, uninfected targets expressing highMHC-I concentrations are likely to occur in the vicinity ofvirus-infected cells due to local IFN-γ release by T cellsinteracting with the cognate target cells in vivo. Uninfectedhigh MHC-expressing cells would be susceptible to lysis orcytokine damage via the cross-reactive activity of low affinityself-reactive CD8+ and CD4+ clones, respectively, foundwithin the ‘virus-specific’ T cell population. Clearly, thiswould result in the destruction of uninfected tissue; in virusencephalitis, such immunopathological damage wouldexacerbate the encephalitic syndrome (Fig. 2). Certainly, theseverity of pathology is a function of the range of TcRspecificities in viral encephalitis, while a restricted range ofTcR specificities is associated with reduced pathology.76 Inour murine model of WNV encephalitis, significant infil-tration of leukocytes into the brain and clinical signs of

214 NJC King and AM Kesson

encephalitis, are reduced in the absence of IFN-γ, withsignificantly increased survival, while neutralizing immunityis effectively established. This suggests that the presence ofIFN-γ may be responsible for the pathogenesis of disease,presumably as a result of an over-vigorous immune response(Shrestha B et al. unpubl. obss). Cross-reactive damage,whether CTL- or cytokine-mediated, is more likely to occurto cells like astrocytes and microglia, i.e. the supporting cellsof the brain, than neurones and IFN-γ and other cytokinesrapidly up-regulate MHC and adhesion molecules on thesecells to high levels. In genetically susceptible individuals, ifthe normal suppression and abrogation of an antiviralresponse failed to occur once the virus was eradicated, thecontinued propagation of self-reactive clones could lead toautoimmune disease,38,70 the infrequency of which is presum-ably testimony to the tight controls which prevent it.

Conclusion

While mechanisms are as yet unclear, flavivirus-mediated up-regulation of cell surface molecules emphasizes the specificeffect of this virus on the target molecules for CTL recogni-tion. Much work has shown that antiviral T cell responsesmay be ‘self ’ cross-reactive. However, we believe this is thefirst instance noted, where a virus may directly drive theimmune response towards the generation of low affinity ‘self ’cross-reactive effector lymphocytes.

This outcome may decoy the immune system into statisti-cally recognizing G0 infected cells with high cell surfaceMHC concentrations in preference to infected cycling cellswith relatively low MHC. The latter produce more virus, anddo not up-regulate MHC and adhesion molecules to the sameextent. These cells may thus escape detection until significantnumbers of high affinity antiviral CTL are produced later on.This would gain more time for a continuing viraemia, whichin turn would increase the chances of virus transmission viahaematophagous arthropods vectors. This could occur withoutthe demise of the vertebrate host which would become immuneagainst further infection. Whether this scenario occurs withhigh efficiency in the current reservoirs for these viruses, inwhich prolonged viraemias are common, is not known. Withthe recent massive increase in interest in these viruses, anumber of lines of investigation will provide further experi-mental data that may confirm or exclude this hypothesis.

Acknowledgements

This work has been supported by Project grants from theNational Health and Medical Research Council, the Austral-ian Research Council Small Grants Scheme, the Watson-Munroe Bequest, the Medical foundation of the University ofSydney and Children’s Hospital at Westmead Fund.

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