Advances in the biology, diagnosis and host-pathogen interactions of parvovirus B19

Jou nal o Medical Mic obiology (2004), 53, 1–17 DOI 10.1099/jmm.0.05485-0 05485 &2004 SGM P in ed in G ea B i ain 1 Re iew Co espondence Amanda Co co an [email p o ec ed] Ad ances in he biology, diagnosis and hos –pa hogen in e ac ions o pa o i us B19 Amanda Co co an and Sean Doyle Na ional Ins i u e o Cellula Bio echnology, Depa men o Biology, Na ional Uni e si y o I eland Maynoo h, Maynoo h, Co. Kilda e, I eland Inc eased ecogni ion o pa o i us B19 (B19), an e y h o i us, as a signi ican human pa hogen ha causes e al loss and se e e disease in immunocomp omised pa ien s has esul ed in in ensi e e o s o unde s and he pa hogenesis o B19- ela ed disease, o imp o e diagnos ic s a egy ha is deployed o de ec B19 in ec ion and blood-p oduc con amina ion and, inally, o elucida e he na u e o he cellula immune esponse ha is elici ed by he i us in di e se pa ien coho s. I is becoming clea ha a leas h ee ela ed e y h o i us s ains (B19, A6/K71 andV9) a e ci cula ing in he gene al popula ion and ha i al en y in o a ge cells is media ed by an expanding ange o cellula ecep o s, including P an igen and -in eg ins. Pe sis en in ec ion by B19 is eme ging as a con ibu o y ac o in au oimmune disease, a hypo hesis ha is cons ained by he de ec ion o B19 in he skin o appa en ly heal hy indi iduals. B19 in ec ion du ing p egnancy may accoun o housands o incidences o e al loss pe annum in Eu ope, No h Ame ica and beyond, ye he e is cu en ly only minimal sc eening o p egnan women o assess se ological s a us, and he eby isk o in ec ion, upon becoming p egnan . Whils majo ad ances in diagnosis o B19 in ec ion ha e aken place, including s anda diza ion o se ological and DNA-based de ec ion me hodologies, blood dona ions ha a e a ge ed a high- isk g oups a e only beginning o be sc eened o B19 IgG and DNA as a means o minimizing exposu e o a - isk pa ien s o he i us. I is now i mly es ablished ha a Th1-media ed cellula immune esponse is moun ed in immunocompe en indi iduals, a inding ha should con ibu e o he de elopmen o an e ec i e accine o p e en B19 in ec ion in selec ed high- isk g oups, including sickle-cell anaemics. Pa o i us B19 In 1975, Y onne Cossa disco e ed wha was o become known as human pa o i us B19 (B19) (Cossa e al., 1975). B19 was i s associa ed wi h disease in 1981, when i was linked o an aplas ic c isis in a pa ien wi h sickle-cell disease. I has since been shown o cause e y hema in ec iosum (EI) ( i h disease o childhood), spon aneous abo ion and some o ms o acu e a h i is (Ande son e al., 1983; Kinney e al., 1988; Wool & Cohen, 1995). B19 is a small, non-en eloped, ssDNA i us and, like all pa o i uses, he capsid p o eins a e a anged wi h icosahe- d al symme y. B19 is 20–25 nm in diame e and has a genome o 5.6 kb (Clewley, 1984; Co mo e & Ta e sall, 1984). The B19 capsid consis s o an 83 kDa mino s uc u al p o ein, VP1, and a 5 kDa majo s uc u al p o ein, VP2. VP2 makes up abou 95 % o he o al capsid, wi h VP1 accoun ing o he emaining 5 % (Ozawa e al., 1987). The sequences o he wo p o eins a e collinea , wi h VP2 being iden ical o he ca boxyl- e minus o VP1; howe e , VP1 comp ises an addi ional 227 aa domain ha is unique o he amino- e minal (Fig. 1). To he le o hese sequences on he B19 genome is he ORF o a non-s uc u al p o ein, NS1, which encodes a p o ein p oduc o 77 kDa. NS1 is a phosphop o ein wi h impo an egula o y unc ions, in- cluding con ol o ansc ip ion (Momoeda e al., 1994a) and i us eplica ion; i also plays a ole in hos -cell dea h (Ozawa e al., 1988). I possesses DNA-binding p ope ies (Raab e al., 2002) and biochemical ac i i ies, such as ATPase, helicase and si e-speci ic endonuclease ac i i ies, as well as nuclea localiza ion signals (Li & Rhode, 1990; McCa y e al., 1992; Jindal e al., 1994; B own & Young, 1997). B19 NS1 has also been shown o a ec G1, bu no G2, a es in e y h oid UT7/Epo-S1 cells (Mo i a e al., 2003). B19 uses a single p omo e , p6, which is capable o exp essing s uc u al and non-s uc u al genes di e en ially (Blundell e al., 1987; A icle numbe = 05485 Doc opic is: e iew Abb e ia ions: B19, pa o i us B19; EI, e y hema in ec iosum; EIA, enzyme immunoassay; FDA, US Food and D ug Adminis a ion; HIV, human immunode iciency i us; HLA, human leukocy e an igen; HSV, he pes simplex i us;IFA,immuno luo escen assay;IFN, in e e on; IL, in e leukin; IUFD, in au e ine e al dea h; IVIG, in a enous immunoglobulin; NIHF, non-immune e al hyd ops; PBMCs, pe iphe al blood mononuclea cells; RA, heuma oid a h i is; RBC, ed blood cell; SI, s imula ion index; TAC, ansien aplas ic c isis; Th cell, T helpe cell; TNF, umou nec osis ac o ; WHO, Wo ld Heal h O ganiza ion. Ozawa e al., 1987). I has been demons a ed ha NS1 in e ac s di ec ly wi h he p6 p omo e and wi h cellula ansc ip ion ac o s Sp1/Sp3 o a ec ansc ip ional eg- ula ion (Raab e al., 2002). Two o he small polypep ides ha e been iden i ied, one encoded by a egion in he middle o he genome wi h a p edic ed M o 7.5 kDa and he o he , which is encoded a he ex eme igh -hand end o he genome, wi h a p edic ed M o 11 kDa (S Amand e al., 1991). Spliced ansc ip s o hese wo polypep ides ha e been ound in in ec ed cells, bu hei unc ion has been hi he o unknown (Luo & As ell, 1993). As a esul o inc eased awa eness o and sc eening o B19, a numbe o no el geno ypes ha e been iden i ied. Se an e al. (2002) sugges ed ha B19 should be classi ied as a geno ype 1 e y h o i us, wi h newly iden i ied s ains A6 (Nguyen e al., 2002) and K71 (Hokyna e al., 2002) classi ied as geno ype 2 and e y h o i us V9 (Nguyen e al., 1998) as he p o o ype o geno ype 3. V9 has o e all nucleo ide sequence a ia ion o abou 12 % om B19 isola es, wi h he majo i y o sequence di e gence occu ing in he 59po ion o he VP1 unique egion; howe e , di e ences in sequence a e no es ic ed o his a ea, bu a e sca e ed h oughou he genome (Nguyen e al., 1999; Heegaa d e al., 2001). K71 pe sis s in human skin and has a nucleo ide di e gence o 10.8 % om B19 and 8.6 % om V9 (Hokyna e al., 2002). In ec i i y, ansmission and epidemiology The only known hos o B19 is humans. The i us eplica es in human e y h oid p ogeni o cells (la e e y h oid cell p ecu so s and bu s - o ming e y h oid p ogeni o s) o he bone ma ow and blood, inhibi ing e y h opoiesis (Mo ime e al., 1983). T opism o p oduc i e B19 in ec ion is mainly due o he es ic i e cellula dis ibu ion o he P blood g oup an igen globoside (Gb4) (B own e al., 1993, 1994), which is ound mos commonly on cells o he e y h oid lineage, bu also on pla ele s, issues om he hea , li e , lung, kidney and endo helium and on syno ium (Cooling e al., 1995; Jo dan & DeLoia, 1999). Indi iduals who lack e y h ocy e P an igens a e e y a e (1 in 200 000) and appa en ly canno be in ec ed by B19 (B own e al., 1994; Chipman e al., 1996). The limi ed opism o B19 is no ully unde s ood, as educed capsid exp ession has been obse ed in non- pe missi e cells; howe e , in acellula ac o s ha a e ound only in e y h oid cells a e assumed o be essen ial o op imal ansc ip ion and i us eplica ion (Ozawa e al., 1987; Ku pad e al., 1999; Gallinella e al., 2000). I has been shown ha he le el o P an igen exp ession on cells is no ela ed di ec ly o he e iciency o i us binding. In addi ion, some cell lines canno be ansduced by a B19 ec o , despi e P an igen exp ession and i us binding o he P an igen, hus indica ing ha a co- ecep o is p obably essen ial o i us en y in o human cells (Weigel-Kelley e al., 2001). Thus, ecen e idence sugges s ha he p esence o he P an igen alone is no su icien o gain en y in o cells (Weigel-Kelley e al., 2001) and i has been sugges ed ha mul iple - in eg ins may unc ion as co- ecep o s o B19 cellula assimila ion (Weigel-Kelley e al., 2003). B19 s udies ha e been hampe ed by di icul ies in p opaga - ing he i us in i o. Recen s udies ha e indica ed ha in ec ion unde hypoxic condi ions [1 % ( / ) O2] causes up egula ion o B19 exp ession, which is associa ed wi h inc eased i us eplica ion and p oduc ion o in ec ious i ions (Pille e al., 2002). Due o he cy o oxic na u e o he non-s uc u al p o ein NS1, no con inuous cell line ha p opaga es B19 has been es ablished (Ozawa e al., 1987; Momoeda e al., 1994b). B19 ansmission occu s mos commonly by pe sonal con- ac ia ae osol o espi a o y sec e ions; howe e , con ami- na ed blood p oduc s, such as clo ing ac o concen a es, a e a sou ce o ia ogenic ansmission (Ande son e al., 1985; Lyon e al., 1989; Williams e al., 1990; San agos ino e al., 1994; E dman e al., 1997). B19 can be ansmi ed ans- placen ally om an in ec ed mo he o he e us, which may lead o non-immune e al hyd ops (NIHF), spon aneous abo ion o in au e ine e al dea h (IUFD) (Clewley e al., 1987; Mille e al., 1998; Skjoldeb and-Spa e e al., 2000). The P blood g oup an igen, which se es as a ecep o o B19, has been de ec ed on cells o he illous ophoblas o placen al issues in a ying amoun s du ing he cou se o p egnancy. In he i s imes e , le els o he P an igen a e e y high; hey begin o decline in he second imes e and become unde ec able by he mid-s ages o imes e 3 (Jo dan & DeLoia, 1999). This high le el o globoside ecep o on placen al cells in ea ly p egnancy may ac as a pa hway o B19 o be ansmi ed om he mo he o he e us, whe eby he i us can hen in ec e y h oid p ogeni o cells o eplica ion. In suppo o his hypo hesis, Wegne & Jo dan (2002) ha e shown conclusi ely ha 125I-labelled VP2 capsid in e ac ion wi h illous cy o ophoblas cells is media ed by P an igen. In ec ion wi h B19 is e y common and cases o in ec ion ha e been epo ed all o e he wo ld in all seasons. Se op e alence inc eases wi h age and, by adul hood, Fig. 1. Schema ic ep esen a ion o human pa o i us B19 genome and ansc ip ion map [ ed awn wi h pe mission om G ay e al . (1998)]. A. Co co an and S. Doyle 2 Jou nal o Medical Mic obiology 53 .70 % o he adul popula ion is se oposi i e (Ke e al., 1999). Child en a e he main sou ce o ansmission and ou b eaks can pe sis o mon hs in schools and day-ca e cen es, due o he ela i ely la ge numbe o se onega i e child en and close con ac o child en wi hin his en i on- men (Tucke man e al., 1986; G illi e al., 1989). The annual se ocon e sion a e among women o childbea ing age has been es ima ed o be 1.5 % du ing endemic pe iods and 13 % du ing epidemics (Koch & Adle , 1989; Valeu -Jensen e al., 1999). Fu he mo e, in ec ion by B19 du ing p egnancy can lead o spon aneous abo ion o e al anaemia. Conse- quen ly, he ques ion mus now be posed whe he he B19 immune s a us o p egnan women should be de e - mined ou inely on ini ial p esen a ion, o acili a e im- p o ed p egnancy ou come wi h espec o po en ial B19 in ec ion. Mani es a ions and clinical symp oms B19 has been associa ed wi h an expanding ange o clinical diso de s since he disco e y ha i is he ae iological agen o EI. This is a mild childhood illness ha is cha ac e ized by an e y hema ous ash ha a ec s he ace, unk and limbs o he body. I is also associa ed wi h complica ions du ing p eg- nancy, acu e a h opa hy, disease in immunocomp omised pa ien s and ansien aplas ic c isis (TAC). B19 in ec ion and p egnancy Exposu e o and in ec ion by B19 can lead o se ious complica ions du ing p egnancy. In ec ion du ing p eg- nancy may esul in e al anaemia, spon aneous abo ion and hyd ops e alis (B own e al., 1984; Kinney e al., 1988; Heegaa d & Ho nsle h, 1995). Abou 30–40 % o women a e non-immune and do no possess neu alizing an ibodies o B19 and, he e o e, a e suscep ible o in ec ion by his i us. A e ical ansmission a e o 33 % has been epo ed by he Public Heal h Labo a o y Se ice in he UK (PHLS, 1990) and o he s ha e epo ed simila a es (B own e al., 1984; Hall e al., 1990), al hough a ecen s udy epo ed a ansmission a e o 51 % (Yaegashi, 2000). The e a e o e ou million bi hs in Eu ope pe annum (Eu os a , 1998) and, because 30 % o p egnan women a e B19- se onega i e, o e 1 200 000 Eu opean women a e he e o e suscep ible o B19 in ec ion du ing p egnancy. Assuming a combined a e o in ec ion and e al loss o 0.2 % (Le y e al., 1997; Mille e al., 1998; Wa e e al., 1998), i can be es ima ed ha app oxima ely 3000 p egnancies pe annum may be los . Analogous bi h a es in he USA and Canada imply ha a simila incidence o e al dea h due o B19 in ec ion can be expec ed o hese coun ies. These calcula ions a e based solely on li e bi hs and, as he numbe o ac ual p egnancies is much highe , he abo e es ima e is conse a i e. P egnan women a e mos suscep- ible o B19 in ec ion du ing epidemics and also when exposed o in ec ed child en in he home (Valeu -Jensen e al., 1999). Du ing ou b eaks, ansmission a es o 25 % in schools and 50 % a home ha e been epo ed (Ande son e al., 1990). Mos p egnan women a e asymp oma ic, bu some do expe ience symp oms, such as exan hema and a h algia (Komischke e al., 1997). As hese symp oms a e commonly associa ed wi h p egnancy, acu e B19 in ec ion can o en be o e looked; howe e , ou ine sc eening o symp oms o B19 in ec ion o se ocon e sion would o e - come his p oblem. Fe al dea h usually occu s 4–6 weeks pos -in ec ion, bu has been epo ed up o 12 weeks a e B19 symp oma ic in ec ion (Hed ick, 1996). A s udy o 427 p egnan women wi h B19 in ec ion in he UK obse ed ha e al loss was con ined o he i s 20 weeks o ges a ion (Mille e al., 1998). This is suppo ed by igu es eleased in he UK and o he s udies, which epo ed ha e al loss as a consequence o in au e ine B19 in ec ion is highes in, bu no es ic ed o, he i s 20 weeks o ges a ion (Hall e al., 1990; PHLS, 1990). The c i ical ime o in ec ion has since been na owed down o he 16 h week o ges a ion (Yaegashi e al., 1999). Mos cases o e al loss due o B19 in ec ion ha e been epo ed in he second imes e (Ende s & Bibe , 1990; To ok, 1990; Wa e e al., 1998). This suscep ibili y could be a ibu ed, a leas in pa , o he ela i e imma u i y o he e al immune esponse a his s age. Mo e impo an , hough, is he opism ha B19 has o e y h oid p ogeni o cells (Yaegashi, 2000) and he ac ha in he second imes e o p egnancy, he li e-span o e al ed blood cells (RBCs) is sho ened and RBC mass inc eases h ee- o ou old du ing his pe iod o ges a ion (Rodis e al., 1988). B19 eplica ion wi hin e y h oid p ogeni o cells leads o apop- osis, which ul ima ely esul s in inhibi ion o e y h opoiesis (Mo ey e al., 1993). E y h oblas openia can hen occu as a consequence o B19 eplica ion, causing se e e e al anaemia. Anaemia is an unde lying ac o in he de elopmen o hyd ops. Fe al hyd ops was i s associa ed wi h B19 in 1984 (B own e al., 1984). Since hen, 10–20 % o NIHF cases ha e been epo ed o be B19-associa ed (Yaegashi e al., 1994; Jo dan, 1996) and, in a s udy o B19 in ec ion in Japanese women du ing p egnancy, he isk o hyd ops was de e - mined o be abou 10 % (Yaegashi e al., 1999). NIHF usually occu s 2–4 weeks a e ma e nal B19 in ec ion (Komischke e al., 1997). Cases o IUFD ha a e associa ed wi h e al hyd ops and caused by B19 ha e been epo ed mos commonly in he second imes e and, o a lesse ex en (unquan i ied as ye ), in he hi d imes e o p egnancy (Sanghi e al., 1997). When cases o IUFD ha occu ed du ing an 18 mon h pe iod in he UK we e examined, i was disco e ed ha 11 dea hs we e caused by B19 in he second imes e and, o hese, only h ee we e hyd opic (W igh e al., 1996). In a sepa a e s udy o e a 16 yea pe iod, en cases o IUFD we e epo ed, which p esen ed in ges a ional weeks 15–29. O hose cases, 90 % o he e uses we e hyd opic, 30–40 % had associa ed hea ailu e and h ee o he ma e nal in ec ions we e asymp oma ic (Mo ey e al., 1993). Un il ecen ly, hi d- imes e e al loss o IUFD caused by acu e B19 in ec ion had no been widely epo ed. Howe e , Pa o i us B19 e iew h p://jmm.sgmjou nals.o g 3 o 93 IUFD cases ha we e examined, 7.5 % had B19 DNA in placen al issue in he absence o e al hyd ops (Skjoldeb and-Spa e e al., 2000). Unusually, none o he in ec ed p egnan women in his s udy showed any clinical symp oms o B19 in ec ion. B19-associa ed IUFD in he inal s ages o ges a ion may ha e been o e looked p e iously, due o inadequa e diagnos ic p ocedu es and he di e ence in clinical ea u es o hi d- imes e B19 in ec ion. The mos s iking obse a ion in hese IUFD cases was he lack o e al hyd ops and he ac ha many o he cases had ei he delayed o absen B19 IgG esponses. His opa hological examina ion o he e uses e ealed no majo abno mali ies. Simila epo s o non-hyd opic, hi d- imes e IUFD ha was associa ed wi h B19 in ec ion ha e been published (Tol ens am e al., 2001a). He e, i was e ealed by PCR analysis o e al o placen al issues ha 15 % o IUFD cases we e a ibu able o B19 in ec ion. This s udy also obse ed delayed B19-speci ic an ibody esponses, as he mo he s in ol ed had no se ological e idence o an acu e B19 in ec ion. Howe e , ollow-up s udies showed e idence o se ocon e sion wi hin 6 mon hs. Tissue samples showed no signs o i us inclusions and immunochemis y analysis e ealed no e idence o B19 p o eins (Tol ens am e al., 2001a). Al hough he concep o B19-induced, hi d- imes e e al loss has p o ed somewha con o e sial (C owley e al., 2001; Sebi e, 2001), i u he illus a es he equi emen s o awa eness o B19 pa hogenesis and diag- nos ic B19 PCR sc eening du ing p egnancy. Fu he mo e, Nunoue e al. (2002) sugges ed s ongly ha p ospec i e s udies o e alua e he ela ionship be ween ime o in ec ion and IUFD, wi h and wi hou signs o e al hyd ops, a e necessa y. In ac , B19 PCR may be he mos sensi i e way o diagnosing in au e ine B19 in ec ion, especially as .50 % o in ec ed e uses es nega i e o B19 IgM (Dieck e al., 1999). Adminis a ion o high- i e in a enous immunoglobulin (IVIG) has been shown o be success ul in ea ing e al hyd ops in some cases (Selbing e al., 1995; Alge , 1997). Fo cases o e al in ec ion, in au e ine blood ans usions may be bene icial (Schwa z e al., 1988a; Hansmann e al., 1989), especially in he case o hyd ops, bu his p ocedu e does in ol e addi ional isks o he ou come o p egnancy (Be y e al., 1992; Came on e al., 1997; Bousque e al., 2000). A s udy by Wa e e al. (1998) epo ed wo cases whe e in au e ine blood ans usions led o he cessa ion o symp oms and he bi h o no mal babies. In a sepa a e s udy, 38 cases o B19-associa ed e al hyd ops we e epo ed, 12 o which ecei ed in au e ine blood ans usion. Al hough h ee o hese e uses subsequen ly died, he p obabili y o dea h among e uses ha did no ecei e a blood ans usion was signi ican ly highe (Fai ley e al., 1995). In addi ion, spon aneous esolu ion o hyd ops wi hou in e en ion has been epo ed, sugges ing ha ea men is no always necessa y (P yde e al., 1992). B19 in ec ion du ing p egnancy is no a signi ican cause o bi h de ec s; howe e , a leas one incidence o congeni al ca diomyopa hy has been linked o B19 in ec ion (Ba on e al., 1997). A h opa hy Like he ubella i us (Lee, 1960), B19 in ec ion has also been linked o a h i is and a h algias, mos commonly in adul s, bu also in child en (Reid e al., 1985). On a e age, 50 % o adul cases o EI ha e associa ed join mani es a ions ha may pe sis o up o 1 mon h (Cassino i e al., 1995). B19 a h i is is usually symme ical, a ec ing mainly he small join s o he hands, w is s and knees (Reid e al., 1985). I is mo e common in emales han males, wi h an es ima ed 60 % o women wi h symp oma ic disease ha mani es s in a h opa hy (Whi e e al., 1985; Wool e al., 1989). Symp- oms gene ally subside wi hin 3 weeks wi hou any damage o he join s (Wool e al., 1991), bu abou 20 % o a ec ed women su e pe sis en o ecu ing a h opa hy. Abou 75 % o hese pa ien s ha e an associa ed ash and ,20 % ha e he ypical ‘slapped cheeks’ acial exan hem. B19 has been p oposed as he causa i e agen o a h i ic condi ions ha exhibi simila symp omologies o hose ound in heuma oid a h i is (RA), ju enile RA and e osi e poly- a h i is, as ecen B19 in ec ion and high le els o B19 an ibodies ha e been e iden in many o hese pa ien s (Whi e e al., 1985; Noc on e al., 1993; Mimo i e al., 1994; Tyndall e al., 1994). I has been sugges ed ha B19- associa ed a h i is is ela ed o ce ain human leukocy e an igen (HLA) haplo ypes o pa ien s, wi h indi iduals o ei he HLA DR4 o B27 being mos suscep ible (Klouda e al., 1986; Jawad, 1993); howe e , i is unclea how B19 causes symp oms ha a e associa ed wi h a h i is. Analogous o he appea ance o exan hema in EI, a h i is usually occu s a e de elopmen o B19-speci ic an ibodies. This sugges s ha symp oms may be due o o ma ion o immune complexes. Despi e he ac ha he P an igen is exp essed on syno ium, i has been shown ha syno ial memb ane cells a e non- pe missi e o B19 (Miki & Chan le , 1992; Cooling e al., 1995). No mal human syno ial ib oblas s ha e been shown o exhibi inc eased in asi eness ollowing exposu e o B19 i aemic se um, as judged by he acqui ed abili y o deg ade econs i u ed ca ilage ma ix (Ray e al., 2001). B19 may gain en y o cells ha possess he B19 ecep o bu a e no ac i ely di iding, esul ing in he p oduc ion o excessi e cy o oxic NS1 (Ozawa e al., 1988). The B19 NS1 p o ein causes he sec e ion o p oin lamma o y cy okines, which could cause he in lamma ion and cell damage ha a e seen in pa ien s wi h B19-associa ed a h i is and o he in lam- ma o y and au oimmune diso de s ha ha e been linked o B19 in ec ions (Mo a e al., 1996; Mi chell, 2002). In one s udy, an ibodies ha we e speci ic o he non-s uc u al p o ein NS1 we e ound in pa ien s wi h pe sis en B19- associa ed a h opa hy, bu no in se um om indi iduals wi h e idence o pas in ec ion wi hou complica ions ( on Poblo zki e al., 1995a), hus sugges ing an al e ed hos esponse in he o me coho . Howe e , o he s ha e dispu ed his, epo ing simila NS1 an ibody eac i i y in A. Co co an and S. Doyle 4 Jou nal o Medical Mic obiology 53 pa ien s wi h ch onic o acu e B19-associa ed a h opa hy (Mi chell e al., 2001) and ecen ly in ec ed heal hy indi i- duals (Sea le e al., 1998; Ennis e al., 2001; Mi chell e al., 2001; Heegaa d e al., 2002a). on Landenbe g e al. (2003) u he sugges ed ha B19 may be in ol ed di ec ly in he induc ion o au oimmune eac ions ha a e media ed, a leas in pa , by an i-phospholipid an ibodies, because o he p e alence o hese an ibodies in pe sis en ly B19-in ec ed indi iduals. The e is signi ican e idence o B19 DNA pe sis ence in bone ma ow, pe iphe al blood and syno ial issues o pa ien s wi h ch onic, B19-associa ed a h opa hy (Fo o e al., 1993; Musiani e al., 1995; Nikka i e al., 1995). Howe e , i has also been shown ha al hough B19 DNA pe sis ed in he syno ium issue o 28 % o child en who p esen ed wi h ch onic a h i is, an e en highe p opo ion (48 %) o se oposi i e, immunocompe en olun ee s had B19 DNA in hei syno ium issues. These esul s imply ha B19 DNA in syno ium issue may no be associa ed di ec ly wi h symp oms o ch onic a h opa hy. None o he indi iduals es ed had e idence o B19 DNA in hei syno ial luid, bone ma ow o blood and all we e posi i e o B19 IgG an ibodies (So ¨de lund e al., 1997). None heless, a ecen epo u he enhanced he co ela ion be ween B19 in ec ion and heu- ma ic childhood disease (Lehmann e al., 2003). This wo k clea ly elucida ed a signi ican di e ence in se um and/o syno ial luid-de i ed B19 DNA (P,0.0001) be ween con ol (9/124, 7 %) and pa ien (26/74, 35 %) specimens and concluded ha he a e o pe sis en B19 in ec ion in hese pa ien s is signi ican ly highe han in age-ma ched con ols. The ecen inding o B19 DNA in 64 % (14/22) o con ol skin biopsies, compa ed o 50 % (18/36) o ch onic u ica ia pa ien s, con i ms ha cau ion should be exe cised in d awing conclusions ega ding B19 in ol emen in skin diso de s and possibly in o he B19-associa ed clinical diso de s (Vuo inen e al., 2002). Ch onic B19 in ec ion in he immunocomp omised hos A hos wi h a comp omised immune sys em is pa icula ly a isk o B19 in ec ion, including people wi h AIDS, cance pa ien s who a e ecei ing chemo he apy and ansplan pa ien s on immunosupp essi e d ugs (Young, 1996). Many a e unable o p oduce neu alizing an ibodies o clea he i us and his can lead o pe sis en in ec ion, esul ing in anaemia (Ku zman e al., 1989a; Young, 1996). In one case s udy, an AIDS pa ien de eloped se e e anaemia as a esul o ch onic pu e ed-cell aplasia ha was caused by B19 in ec ion (Kodu i e al., 1997). Despi e emission ollowing IVIG ans usions, he pa ien su e ed se e al ecu ences o se e e anaemia. In ano he epo , se a ob ained om ansplan pa ien s who we e ecei ing bone-ma ow g a s (n¼27) we e analysed by PCR (Schleuning e al., 1999). O he coho es ed, 15 % we e B19 DNA-posi i e and many o hese pa ien s also de eloped a e icula ash. Al hough de elopmen o a ash du ing B19 in ec ion is hough o be media ed by o ma ion o immune complexes, hese pa- ien s did no exhibi any signs o a B19-speci ic an ibody esponse. The e o e, i was hypo hesized ha he ash was a consequence o a di ec i us e ec on he skin; his inding is suppo ed by he ac ha B19 DNA has been ound p e iously in a skin biopsy om a male pa ien wi h B19- associa ed e e , ash and polya h i is (Nikka i e al., 1996). Pe sis an B19 in ec ion esul s in ch onic supp ession o e y h opoiesis wi h ch onic anaemia. A epo by G ae e e al. (1989) desc ibed how ou child en who we e unde - going cance chemo he apy ea men we e in ec ed by B19, esul ing in ch onic bone-ma ow supp ession. Schleuning e al. (1999) also epo ed ha one o he ansplan pa ien s subsequen ly died om hea ailu e and B19 DNA was de ec ed in he myoca dium, bu no in pe iphe al blood, indica ing ha hea ailu e was a consequence o B19 in ec ion [hea ailu e has been ecognized as a ea u e o B19 in ec ion in he pas (Chia & Jackson, 1996)]. Ano he o he ansplan pa ien s in es iga ed was ound o ha e de eloped hepa i is (Schleuning e al., 1999), which was also a ibu ed o B19 in ec ion (Yo o e al., 1996). As hese ansplan pa ien s we e subjec ed o s ic decon amina ion p ocedu es, including isola ion in single ooms wi h posi- i e ai low and decon amina ed ood, i is hus unlikely hey con ac ed B19 ia espi a o y sec e ions. Pla ele concen a es we e sc eened o B19 p io o adminis a ion and we e he e o e no a likely sou ce o ansmission. Howe e , as B19 DNA in clo ing and immunoglobulin concen a es is known o cause in ec ion, his may ha e been he ou e o ansmission o he i us (Saldanha & Mino , 1996). Many immunocomp omised pa ien s wi h ch onic anaemia espond posi i ely o IVIG he apy; howe e , indi iduals may su e om ecu en elapses o aplasia (Kodu i e al., 1997, 1999; Moudgil e al., 1997). In addi ion, adminis a ion o IVIG may no always be e ec i e, as in ec ion may pe sis despi e ea men , pa icula ly in ansplan pa ien s who a e hea ily immunosupp essed (Moudgil e al., 1997; Schleuning e al., 1999; Lui e al., 2001). To da e, no da a a e a ailable on he ac ual p o ec i e le el o B19 IgG, al hough le els o .6IUml 1a e hough o be p o ec i e (Sea le e al., 1997). As pa ien s ail o moun an an ibody esponse, se ological diagnosis is u ile and de ec ion o B19 in ec ion is he e o e usually achie ed by B19 DNA de ec ion ia a PCR assay. T ansien aplas ic c isis (TAC) B19-associa ed TAC may occu in indi iduals who exhibi unde lying ch onic haemoly ic diso de s, such as he edi a y sphe ocy osis (Beland e al., 1997). In 1981, i was disco e ed ha B19 caused TAC in child en wi h sickle-cell anaemia (Se jean e al., 1981) and i is now clea om subsequen s udies ha almos 70 % (118/177) o B19 in ec ions in his coho esul ed in TAC (Se jean e al., 2001). Pa o i us B19 e iew h p://jmm.sgmjou nals.o g 5 Vaccine No speci ic he apy is equi ed o B19 in ec ion in immu- nocompe en indi iduals. Symp oms o a h opa hy can be ea ed wi h non-s e oidal, an i-in lamma o y d ugs. Humo al immune esponse B19 i aemia occu s 1 week a e exposu e and usually las s abou 5 days, wi h i us i es peaking on he i s 2 days. B19- speci ic IgM an ibodies a e de ec ed la e in he i aemic s age (a abou day 10 o 12) and can pe sis o up o 5 mon hs (Ande son e al., 1985; Schwa z e al., 1988b; Yaegashi e al., 1989) bu , in some pa ien s, can las e en longe (Musiani e al., 1995). Speci ic IgG an ibodies a e de ec able abou 15 days pos -in ec ion, emain high o se e al mon hs and pe sis long- e m. IgA an ibodies a e de ec able o a sho pe iod ollowing he onse o clinical symp oms (E dman e al., 1991). De elopmen o he an ibody esponse co e- sponds o i us clea ance and also, in he as majo i y o cases o B19 in ec ion in immunocompe en indi iduals, p o ec ion om disease (Ande son e al., 1985). A s udy o child en wi h sickle-cell disease showed ha hose who had had one episode o B19-associa ed TAC did no su e a second episode (Se jean e al., 1993). His o ically, he B19 VP1 p o ein, and in pa icula he VP1- unique egion, was hough o be he immunodominan an igen and i s inco po a ion in o se ological assays was hough o be essen ial (Raymen e al., 1990). Howe e , i is now clea ha his obse a ion, which was based on he absence o an ibodies o linea epi opes o VP2 when sc eened by Wes e n blo , was somewha e oneous. I has now been es ablished conclusi ely ha an ibodies agains capsid VP2 a e main ained, e en when B19 IgG di ec ed agains he VP1-unique egion is los (Ke e al., 1999; Mana esi e al., 1999; Co co an e al., 2000). Speci ic an i- i us an ibody is conside ed o be a signi ican mechanism o immune p o ec ion, based on he ci cum- s an ial e idence ha high-dose immunoglobulin he apy is some imes bene icial in in ec ed pa ien s (Ku zman e al., 1989b; Schwa z e al., 1990). An ibodies agains linea epi opes o VP2 and, o some ex en , VP1 disappea ab up ly a e B19 in ec ion, whe eas IgG eac i i y agains con o ma- ional epi opes o bo h VP1 and VP2 pe sis s (So ¨de lund e al., 1995; Ke e al., 1999). Pe sis en in ec ions ha a e associa ed wi h ch onic anaemia whe e he immune esponse o B19 has ailed o p oduce neu alizing an ibodies o hey ha e been a e y low le els ha e been obse ed (Ku zman e al., 1987, 1988; Coulombel e al., 1989). Diagnosis o B19 in ec ion – choice o an igen Accu a e labo a o y diagnosis o ecen B19 in ec ion o pas exposu e elies on sc eening plasma specimens o ei he speci ic an ibody eac i i y agains i us capsid p o eins ha a e exp essed in euka yo ic exp ession sys ems (e.g. he baculo i us exp ession sys em) o o B19 DNA by using PCR. Immunoassays ha only inco po a e Esche ichia coli- exp essed B19 an igens, which ha e unde gone dena u a ion as pa o he manu ac u ing p ocess, will p oduce alse- nega i e esul s, due o he absence o con o ma ional epi opes (Jo dan, 2000). A unique ad an age o he euka - yo ic baculo i us exp ession sys em is i s abili y o di ec he pos - ansla ional p o ein olding ha is necessa y o he p oduc ion o soluble, con o ma ionally in ac VP2 capsid p o eins (B own e al., 1990; Ke e al., 1995a). Unlike B19 VP2, VP1 does no appea o o m soluble capsid s uc u es; howe e , VP1 has been p oduced as a ‘con o ma ionally in ac ’ p o ein ha e ains con o ma ional epi opes ha a e p esen in he na i e i ion (B own e al., 1990; Ke e al., 1999). A numbe o au ho s ha e s a ed ha co-exp ession o VP1 and VP2 in euka yo ic exp ession sys ems esul s in he o ma ion o emp y capsids ha a e an igenically analogous o na i e B19 i ions. Fu he mo e, i has been hypo hesized ha such co-capsids con ain con o ma ional epi opes ha a e essen ial o accu a e de ec ion o in ec ion (Kajigaya e al., 1989, 1991; F anssila e al., 2001; Ballou e al., 2003). To da e, he e a e no da a o p o e ha co-capsids a e ac ually p esen in such p epa a ions and i canno be excluded ha sepa a e, con o ma ionally in ac en i ies ha a e comp ised o ei he VP1 o VP2 a e in ac p esen . Mo e ecen e idence om a numbe o au ho s now sugges s ha B19 NS1 IgG and IgM de ec ion also plays a signi ican ole in diagnosis o acu e in ec ion, he eby supplemen ing he ole played by B19 capsid an igens as diagnos ic an igens (Ennis e al., 2001; Heegaa d e al., 2002a). B19 IgM immunode ec ion Acu e B19 in ec ions a e con i med by B19-speci ic IgM eac i i y, whe eas pas in ec ions a e de ec ed by IgG eac i i y (Ande son e al., 1985). In mos si ua ions, IgM an ibodies appea 7–10 days pos -in ec ion and a e di ec ed agains linea and con o ma ional epi opes o VP1 and VP2 (Palme e al., 1996; Mana esi e al., 2001). I has been epo ed ha IgM agains con o ma ional epi opes on VP1 and VP2 and agains linea epi opes on VP1 appea s a he same ime pos -in ec ion and a he same equency. How- e e , i was also e ealed ha IgM eac i i y agains he mino capsid p o ein, VP1, may pe sis somewha longe pos - in ec ion (Palme e al., 1996; Mana esi e al., 2001). I IgM esponses agains con o ma ional VP1 pe sis when o he B19-speci ic IgM an ibodies a e absen , hen diagnos ic echniques ha inco po a e con o ma ional VP1 may no be he mos sui able ma ke s o acu e B19 in ec ion. How- e e , ano he s udy obse ed no di e ence in IgM eac i i y agains con o ma ional epi opes o he capsid p o eins in diagnosing B19 in ec ion (Ke e al., 1999). Fu he mo e, hese au ho s obse ed no dispa i y in IgM eac i i y agains na i e (con o ma ionally in ac ) and linea ized an igens o bo h VP1 and VP2. P esen ly, he e is no In e na ional S anda d p epa a ion o B19 IgM and only one B19 IgM diagnos ic es ha has been A. Co co an and S. Doyle 6 Jou nal o Medical Mic obiology 53 clea ed by he US Food and D ug Adminis a ion (FDA) is a ailable. I is a mì-cap u e enzyme immunoassay (EIA) ha u ilizes B19 ecombinan VP2 capsids o he de ec ion o speci ic IgM in human se um o plasma. This immunoassay has 89.1 % sensi i i y and 99.4 % speci ici y (Doyle e al., 2000) and is used widely o he diagnosis o ecen B19 in ec ion (Jo dan, 2000; Mi chell e al., 2001; Vuo inen e al., 2002). Fu he mo e, alida ed al e a ion o he immuno- assay cu -o , based on ecei e ope a ing cha ac e is ic analysis, acili a es imp o ed immunoassay sensi i i y, which may ha e a u ili y in de ec ion o lowe le els o B19-speci ic IgM in immunocomp omised indi iduals and young child en (Doyle e al., 2000). No e idence o c oss- eac i i y wi h o he i al in ec ions, such as ubella, mumps, a icella-zos e i us, cy omegalo i us, he pes simplex i us- 1 (HSV-1) and HSV-2, is appa en when his immunoassay is used in clinical se ings. P e ious s udies ha e epo ed c oss- eac i i y wi h ubella in se e al comme cial B19 IgM assays (Sloo s & De ine, 1996; Tol ens am e al., 1996) and as he symp oms o ubella in ec ion a e simila o hose o B19 in ec ion, his was a cause o conce n, pa icula ly in he diagnosis o in ec ion in p egnan women. A alse-posi i e a e o 5 % was epo ed when specimens om heal hy olun ee s we e analysed wi h a ange o comme cially a ailable B19 IgM immunoassays, p obably due o c oss- eac i i y and lack o speci ici y in hese immunoassays (Tol ens am e al., 1996). De ec ion o B19 NS1 IgM has ecei ed li le a en ion as a ma ke o ecen in ec ion by B19. Ennis e al. (2001) obse ed ha 27.5 % (11/40) o specimens ha we e B19 VP2 IgM-posi i e also con ained B19 NS1 IgM when es ed by ELISA. In e es ingly, when hese samples we e analysed by Wes e n blo , he e was no e idence o NS1 IgM eac i i y, which indica es ha con o ma ional epi opes a e impo an o de ec ion. B19 IgG immunode ec ion De elopmen o B19 IgG an ibodies coincides wi h a decline in he IgM esponse. IgG eac i i y agains con o ma ional epi opes o VP1 and VP2 pe sis s pos -in ec ion; howe e , o bo h capsid p o eins, eac i i y agains linea epi opes declines pos -in ec ion [ab up ly agains VP2, bu mo e slowly agains VP1 (So ¨de lund e al., 1995; Kaikkonen e al., 1999; Ke e al., 1999; Mana esi e al., 1999)]. An ibody eac i i y agains linea VP2 epi opes usually disappea s wi hin 6 mon hs o B19 in ec ion (So ¨de lund e al., 1995). This ini ial eac i i y agains linea ized VP2 appea s o be di ec ed p edominan ly agains a hep apep ide (amino acids 344–350) ha was iden i ied by analysis o acu e-phase se a (Kaikkonen e al., 1999). Al hough he an ibody esponse wanes agains linea epi- opes on B19 capsid p o eins, i pe sis s agains con o - ma ional epi opes o bo h capsid p o eins. The only FDA-clea ed B19 IgG immunoassay ha is a ailable as a ma ke o pas in ec ion is a mic opla e immunoassay ha u ilizes capsid VP2 o de ec B19 and e y h o i us V9 IgG (Heegaa d e al., 2002b; A. Ga ba g-Chenon, pe sonal communica ion). This baculo i us-based immunoassay has been compa ed o ano he comme cially a ailable, E. coli-based VP1 immunoassay o de ec ion o B19 an ibodies in he se a o p egnan women (Jo dan, 2000). A numbe o equi ocal esul s we e ob ained by using he B19 VP1 immunoassay. To e i y esul s, samples we e es ed by using a comme cially a ailable VP1 immuno luo escen assay (IFA). This assay examines se o eac i i y agains con o ma- ionally in ac VP1. Al hough his assay may be somewha subjec i e, as i p ima ily measu es he deg ee o specimen luo escence, when used in pa allel wi h he VP2 IgG immunoassay, i can con i m B19 eac i i y. Resul s om he VP1 IFA we e simila o hose ob ained by he baculo- i us VP2 EIA and we e in acco dance wi h he ac ha many o he samples ha we e ound o be equi ocal by he E. coli VP1 EIA had clinical his o ies o B19 exposu e. A ailabili y o a B19 IgG In e na ional S anda d (2nd In e na ional S an- da d 2003; code 01/602; 77 IU pe ampoule) should u he assis in accu a e con i ma ion o pas B19 in ec ion by s anda dizing B19 IgG de e mina ion om di e en labo a- o ies ha use a a ie y o es sys ems (Fe guson e al., 1997; Sea le e al., 1997). Recen ly, he impo ance o an ibodies agains he B19 non- s uc u al p o ein NS1 has been in es iga ed, wi h a iew o imp o ing diagnosis o B19 in ec ion. The p esence o B19 NS1 IgG was hough o be associa ed p ima ily wi h pe sis en B19 in ec ion ( on Poblo zki e al., 1995a, 1995b); howe e , se e al g oups ha e subsequen ly ound no signi ican di e ence be ween he le el o NS1 IgG in con ol pa ien s wi h pas in ec ion and hose wi h ch onic B19 in ec ion (Sea le e al., 1998; Ven u oli e al., 1998; Jones e al., 1999). Mapping o B-cell epi opes on NS1 iden i ied h ee an igenic egions (amino acids 191–206, 271–286 and 371–386) ha we e equally eac i e wi h se a om heal hy indi iduals wi h pas B19 in ec ion and pa ien s who we e in ec ed pe sis en ly by B19 (Tol ens am e al., 2000). Mo e ecen ly, by using an E. coli exp ession sys em, NS1 IgG eac i i y has been shown o be mos p e alen in se um, ollowing ecen in ec ion in p egnan women (61 %) (Hemaue e al., 2000). These indings a e suppo ed by he wo k o Mi chell e al. (2001), who examined NS1 IgG eac i i y in se a om indi iduals who we e ei he in ec ed by B19, had been exposed o B19 bu we e no in ec ed, we e su e ing om a ash illness o ch onic a h opa hy, o we e heal hy con ols. NS1 IgG eac i i y was p edominan in ecen ly in ec ed specimens and when ollow-up samples om hese indi iduals we e analysed, he le el o NS1- speci ic IgG eac i i y had declined. In addi ion, he e was no e idence o a connec ion be ween NS1 IgG an ibodies and he de elopmen o a h opa hy (Mi chell e al., 2001). The NS1-speci ic IgG esponse wanes pos -in ec ion as he i us is clea ed om he body; he e o e, NS1 IgG eac i i y may ha e some alue as a ma ke o ecen in ec ion, in conjunc- ion wi h he de ec ion o IgG agains linea epi opes on VP2 (Ennis e al., 2001). This s udy demons a ed ha 69 % o child en who had been in ec ed ecen ly by B19 we e NS1 Pa o i us B19 e iew h p://jmm.sgmjou nals.o g 7 IgG-se oposi i e (Ennis e al., 2001). Heegaa d e al. (2002a) also obse ed a se op e alence o 60 % B19 NS1 IgG in ecen ly in ec ed indi iduals (,6 weeks pos -in ec ion) and sugges ed ha NS1 IgG de ec ion may signi ican ly imp o e immunoassay sensi i i y. I is now clea ha B19 IgM and IgG de ec ion is op imal in immunoassays ha u ilize VP2 capsids o an ibody de ec- ion. An ibody (IgG/M) de ec ion o B19 NS1 p o ein may assis in he con i ma ion o ecen B19 in ec ion, when used in combina ion wi h VP2 capsid-based immunoassays. E y h o i us V9 an ibody de ec ion is also easible, by using immunoassays ha a e based on B19 VP2 capsids. Cell-media ed immuni y Cell-media ed immuni y o B19 has no been s udied ex ensi ely; his is due p ima ily o he ac ha he humo al esponse was hough o be mos impo an in comba ing B19 in ec ion. Indeed, ini ial a emp s o demons a e speci ic T-cell p oli e a i e esponses o B19 we e unsuccess- ul (Ku zman e al., 1989a) and, o some ime, his wo k suppo ed he p e ailing heo y ha neu alizing an ibody p oduc ion was he majo mechanism o immuni y in B19. In 1996, ex i o B19-speci ic CD4þT-cell esponses we e i s de ec ed agains E. coli-exp essed VP1, VP2 and NS1 an igens ( on Poblo zki e al., 1996). T-cell esponses o 16 indi iduals we e analysed ( en se oposi i e and six se onega i e blood dono s), none o whom had any e idence o acu e in ec ion. The majo i y (90 %) o se oposi i e dono s who we e s imula ed ex i o by VP2 displayed speci ic T-cell esponses, wi h 80 % displaying VP1-speci ic esponses. The e was no signi ican di e ence in T-cell p oli e a ion o NS1 be ween se oposi i e and se onega i e indi iduals. Upon inclusion o mAbs ha we e speci ic o class I and class II HLA, i was ound ha HLA class II-speci ic an ibodies inhibi ed T-cell p oli e a ion, hus indica ing ha he e ec o T-cell popu- la ion o B19 a e CD4þcells. Subsequen pe iphe al blood mononuclea cell (PBMC) deple ion o ei he CD4þo CD8þT cells and s imula ion o he emaining popula ion con i med his obse a ion. Mo e ecen ly, signi ican ex i o T-cell eac i i y was ob- se ed in PBMCs o ecen ly and emo ely in ec ed indi i- duals by using a B19 candida e accine (F anssila e al., 2001) and also he B19 ecombinan p o eins, VP1 and VP2 (Co co an e al., 2000). T cells om ecen ly in ec ed indi iduals esponded s ongly o he B19 capsids, gi ing a mean T-cell s imula ion index (SI) o 36 (F anssila e al., 2001). Blood dono s wi h pas in ec ions ga e compa able a es o T-cell s imula ion. Se onega i e indi iduals had SI alues o abou 3.3 and his s udy also showed ha he esponding popula ion o T cells we e CD4þ. Al hough on Poblo zki e al. (1996) saw no di e ence in T-cell esponses o NS1 in se onega i e and se oposi i e indi iduals, signi i- can esponses o his an igen ha e been epo ed in ecen ly in ec ed indi iduals and pa ien s who de eloped ch onic a h opa hy ollowing B19 in ec ion (Mi chell e al., 2001). T-cell esponses o NS1 we e no seen in heal hy indi iduals wi h pas B19 in ec ion, excep o wo indi iduals who we e also NS1 IgG-se oposi i e. Cellula immune esponse o an epi ope o NS1 ha is ecognized speci ically by CD8þT cells was in es iga ed ecen ly by using majo his ocompa ibili y complex e a- me ic complex binding (Tol ens am e al., 2001b). The esponse o 21 indi iduals o his epi ope was examined in heal hy olun ee s and human immunode iciency i us (HIV)-1 in ec ed adul s and child en. Six een o he olun- ee s we e HLA-ma ched (HLA B35) and six we e mis- ma ched. Six y- h ee pe cen o ma ched indi iduals displayed speci ic CD8þT-cell esponses. Se en y- wo pe cen o ma ched indi iduals in he same coho exhibi ed speci ic T-cell esponses by using an in e e on ª(IFN-ª) ELISpo assay. The le el o B19-speci ic CD8þT cells was simila among heal hy and HIV-in ec ed indi iduals. The esul s p esen ed in his epo showed he impo an cellula ole o cy o oxic T cells in comba ing B19 in ec ion (Tol ens am e al., 2001b). B19-speci ic T-cell esponses may now ep esen a no el me hod o con i ming pas B19 in ec ion. Recen e idence shows he impo ance o e alua ing T-cell esponses in unde s anding he na u e o B19 in ec ion. Chen e al. (2001) ha e iden i ied an AIDS pa ien wi h pe sis en B19 in ec ion who showed an ini ial emission o B19 in ec ion. This emission was e iden despi e he lack o a speci ic an ibody esponse, hus indica ing a ole o cellula immuni y in comba ing B19 in ec ion. NS1- eac i e lym- phocy es ha e been de ec ed in wo B19-se onega i e in- di iduals who we e exposed o he i us, indica ing a possible subclinical B19 in ec ion o pe haps a loss o an ibodies agains capsid p o eins (Mi chell e al., 2001). The impo - ance o cellula immuni y in B19 was u he emphasized in a epo by Tol ens am e al. (2001b). He e, in es iga ions o B19-speci ic CD8þT-cell esponses iden i ied wo heal hy adul s and wo HIV-1-in ec ed pa ien s who we e se onega- i e o B19, wi h speci ic T-cell esponses agains B19 by ei he IFN-ªELISpo o e ame binding s udies, hus implying he p esence o a cellula esponse in he absence o a humo al esponse. Signi ican T-cell ansc ip ional ac i a ion has been e- po ed in a pa ien wi h acu e B19 in ec ion, causing in- c eased le els o in e leukin (IL) 1, IL6 and IFN-ªmRNA (Wagne e al., 1995). A subsequen s udy ha analysed he se a o pa ien s who we e in ec ed acu ely by B19 showed ha al hough IL1, IL6, IFN-ªand umou nec osis ac o Æ (TNF-Æ) we e sec e ed du ing he acu e phase o in ec ion, inc eased le els o bo h IFN-ªand TNF-Æpe sis ed and we e de ec able 2–37 mon hs la e , du ing a ollow-up s udy (Ke e al., 2001). I has also been sugges ed ha cy okine gene ic polymo phisms may, in some way, a ec he de elopmen o symp oms du ing B19 in ec ion. To da e, he ans o ming g ow h ac o (TGF) allele has been associa ed wi h skin ash a acu e in ec ion and he IFN-ªallele has been associa ed wi h NS1 an ibody de elopmen (Ke e al., A. Co co an and S. Doyle 8 Jou nal o Medical Mic obiology 53 2003). In a s udy o ecen ly in ec ed child en, i was shown ha al hough s ong T-cell p oli e a i e esponses we e e iden o bo h capsid p o eins, p oduc ion o he T helpe (Th1) cy okine IFN-ª, bu no o IL2, was impai ed when compa ed o con alescen adul s (Co co an e al., 2000). In addi ion, ex i o p oduc ion o IFN-ªand IL2 ha was obse ed in B19-se oposi i e p egnan women was lowe han obse ed p e iously o heal hy, non-p egnan indi i- duals, sugges ing a possible dimuni ion o he ma e nal an i- i us immune esponse ha may subsequen ly inc ease he isk o e al B19 in ec ion (Co co an e al., 2003). Exp ession o he non-s uc u al p o ein NS1 causes he p oduc ion o inc eased le els o he in lamma o y cy okine IL6 in a numbe o cell lines, including hema opoie ic cell lines and human umbilical ein endo helial cells (Mo a e al., 1996). IL6 is known o be in ol ed in syno ial cell p oli e a ion and, in addi ion, high le els o IL6, along wi h o he in lamma o y cy okines, ha e been ound in in lamed join s o pa ien s wi h RA, which would sugges an associa ion be ween IL6 p oduc ion and he join mani es a ions ha a e obse ed wi h B19 in ec ion (Ba aille e al., 1995). IL6 in ol emen in RA is suppo ed by he ac ha an ibodies agains IL6 cause inhibi ion o RA mani es a ions (Ba aille e al., 1995). As well as inc eased IL6 p oduc ion, high le els o IFN-ª, TNF-Æand IL8 ha e been de ec ed in he se a o in an s wi h B19- associa ed acu e myoca di is (Nig o e al., 2000). IL2 p oduc ion a he ma e nal– e al in e ace in women who se ocon e ed o B19 du ing p egnancy is hough o de e mine he ou come o he p egnancy, wi h high le els o IL2 on he e al side being associa ed wi h p egnancies ha esul in a poo ou come (Jo dan e al., 2001). B19 accine Ballou e al. (2003) ha e shown ecen ly ha a ecombinan accine (MEDI-491; Medimmune) ha is comp ised o B19 VP1 and VP2 capsid p o eins could elici neu alizing an i- body i es in olun ee adul s (n¼24). Se a om immu- nized indi iduals we e also shown o be capable o inhibi ing B19 eplica ion in i o. The e icacy o his o mula ion in p e en ing in ec ion by B19 emains o be es ablished; none heless, i is an encou aging and welcome ad ance in he igh agains his ubiqui ous pa hogen. PCR de ec ion o B19 DNA Many clinical labo a o ies now complemen B19 an ibody sc eening wi h diagnos ic PCR and i is well-es ablished ha B19 PCR imp o es he sensi i i y o de ec ion o B19 in ec ion (Skjoldeb and-Spa e e al., 2000; Mana esi e al., 2002). Howe e , cau ion mus be exe cized wi h ega d o he deploymen o B19 PCR o a numbe o easons: (i) he high i aemia ha is associa ed wi h B19 in ec ion, along wi h esul an amplicon load, may cause PCR alse posi i i y – pa icula ly when nes ed PCR is used o B19 de ec ion; (ii) B19 DNA de ec ion may no always be indica i e o an acu e in ec ion; (iii) many PCR assays use in-house p ime pai s o unde ined sensi i i y o de ec ion; (i ) alse nega i i y may be obse ed wi h espec o non-B19 s ains (e.g. e y h o i us V9, K71 o A6), due o mino sequence di e ences (Hokyna e al., 2002; Nguyen e al., 2002; Se an e al., 2002); and, inally, ( ) many ex ac ion me hods a e sui able o DNA pu i ica ion om se um o plasma only and no om solid issue (e.g. placen a o e al issue). No wi hs anding hese ca ea s, some o which a e discussed in mo e de ail below, B19 PCR is an impo an ool in he echnologis ’s a mou y o de ec ion o B19 in ec ion. Du ing acu e in ec ion wi h B19, i al i es can each 1012 genome equi alen s ml1(P owse e al., 1997). In he immunocompe en hos , i us DNA is de ec able o a leas 1 mon h pos -in ec ion (E dman e al., 1991). In ch onic B19 in ec ion, i us DNA can pe sis in he hos wi hou he p esence o B19 IgM o IgG (Ku zman e al., 1988; F ickho en e al., 1990). Howe e , i has also been shown ha B19 DNA can pe sis in heal hy, immunocompe en indi iduals a low le els o long pe iods (Cassino i e al., 1993; Ke e al., 1995b; Musiani e al., 1995; Cassino i & Siegl, 2000). Thus, B19 DNA, de ec ed by quali a i e PCR analysis, is no always indica i e o ecen in ec ion. Cassino i & Siegl (2000) used quan i a i e PCR o ollow he amoun o speci ic B19 DNA in an immunocompe en pa ien , exp essed as genome equi alen s ml1, om he ime o acu e B19 in ec ion un il con alescence. A se ies o samples was aken o e a 1 yea pe iod and was analysed by using a eal- ime luo ogenic PCR assay. Du ing he i aemic s age o B19 in ec ion, i al load eached le els o 8.83109 genome equi alen s (ml blood)1. A his s age, he pa ien was posi i e o speci ic IgM and nega i e o IgG eac i i y. A week 164, i al load had declined o 95 genome equi a- len s ml1, IgM eac i i y was los and con o ma ional IgG eac i i y was s ong. Specimens aken a e his ime-poin we e unde ec able o B19 DNA. Thus, whils he ac ual amoun o ci cula ing B19 DNA ha was p esen ollowing B19 in ec ion diminished d ama ically a e he i s ew weeks o in ec ion, i pe sis ed o some ime be o e being clea ed om he hos , despi e he de elopmen o ci cula ing B19 IgG. This slow a e o B19 DNA clea ance om an immunocompe en hos could impac nega i ely on PCR as a diagnos ic ool in di e en ia ing be ween ecen o ch onic B19 in ec ion in a si ua ion whe e a quali a i e PCR assay o unspeci ied sensi i i y o de ec ion was employed. Howe e , wi h he in oduc ion o he Wo ld Heal h O ganiza ion (WHO) In e na ional S anda d o Pa o i us B19 DNA (NIBSC 99/800), PCR assay s anda diza ion has become possible (Saldanha e al., 2002). Using he WHO s anda d, a compa ible PCR-ELISA ha can de ec le els as low as 1.63103IU B19 DNA ml1was es ablished (Daly e al., 2002) and, by using eal- ime PCR echnology, a sensi i i y o de ec ion o 15.4IUml 1(10 Bax e uni s ml1) (Abe ham e al., 2001) was eached. Mu ¨lle e al. (2002) and Thomas e al. (2003) ha e also desc ibed s anda dized B19 PCR assay sys ems. These s anda dized me hods could be used no only in a diagnos ic se ing, bu also o apid sc eening o plasma mini-pools and blood p oduc s, he eby leading o de e mi- na ion o he amoun o B19 DNA p esen and imp o ed p oduc sa e y. Pa o i us B19 e iew h p://jmm.sgmjou nals.o g 9 Tol ens am, T., Papadogiannakis, N., No beck, O., Pe e sson, K. & B oliden, K. (2001b). F equency o human pa o i us B19 in ec ion in in au e ine e al dea h. Lance 357, 1494–1497. To ok, T. J. (1990). Human pa o i us B19 in ec ions in p egnancy. Pedia In ec Dis J 9, 772–776. Tucke man, J. G., B own, T. & Cohen, B. J. (1986). E y hema in ec iosum in a illage p ima y school: clinical and i ological s udies. J R Coll Gen P ac 36, 267–270. Tyndall, A., Jelk, W. & Hi sch, H. H. (1994). Pa o i us B19 and e osi e polya h i is. Lance 343, 480–481. Valeu -Jensen, A. K., Pede sen, C. B., Wes e gaa d, T., Jensen, I. P., Lebech, M., Ande sen, P. K., Aaby, P., Pede sen, B. N. & Melbye, M. (1999). Risk ac o s o pa o i us B19 in ec ion in p egnancy. JAMA 281, 1099–1105. Ven u oli, S., Gallinella, G., Mana esi, E., Gen ilomi, G., Musiani, M. & Ze bini, M. (1998). IgG esponse o he immuno eac i e egion o pa o i us B19 nons uc u al p o ein by immunoblo assay wi h a ecombinan an igen. J In ec Dis 178, 1826–1829. on Landenbe g, P., Lehmann, H. W., Kno ¨ll, A., Do sch, S. & Mod ow, S. (2003). An iphospholipid an ibodies in pedia ic and adul pa ien s wi h heuma ic disease a e associa ed wi h pa o i us B19 in ec ion. A h i is Rheum 48, 1939–1947. on Poblo zki, A., Gigle , A., Lang, B., Wol , H. & Mod ow, S. (1995a). An ibodies o pa o i us B19 NS-1 p o ein in in ec ed indi iduals. J Gen Vi ol 76, 519–527. on Poblo zki, A., Hemaue , A., Gigle , A., Puchhamme -S ockl, E., Heinz, F. X., Pon , J., Laczika, K., Wol , H. & Mod ow, S. (1995b). An ibodies o he nons uc u al p o ein o pa o i us B19 in pe sis- en ly in ec ed pa ien s: implica ions o pa hogenesis. J In ec Dis 172, 1356–1359. on Poblo zki, A., Ge des, C., Reischl, U., Wol , H. & Mod ow, S. (1996). Lymphop oli e a i e esponses a e in ec ion wi h human pa o i us B19. J Vi ol 70, 7327–7330. Vuo inen, T., Lammin aus a, K., Ko ilainen, P. & Nikka i, S. (2002). P esence o pa o i us B19 DNA in ch onic u ica ic and heal hy human skin. J Clin Vi ol 25, 217–221. Wagne , A. D., Go onzy, J. J., Ma eson, E. L. & Weyland, C. M. (1995). Sys emic monocy e and T-cell ac i a ion in a pa ien wi h human pa o i us B19 in ec ion. Mayo Clin P oc 70, 261–265. Wakama su, C., Takaku a, F., Kojima, E. & 7 o he au ho s (1999). Sc eening o blood dono s o human pa o i us B19 and cha ac e iza- ion o he esul s. Vox Sang 76, 14–21. Wa e, P., Dewilde, A., Sub il, D., And eole i, L. & Thi ion, V. (1998). A clinical and epidemiological s udy o human pa o i us B19 in ec ion in e al hyd ops using PCR Sou he n blo hyb idiza ion and chemilumin- escence de ec ion. J Med Vi ol 54, 140–144. Wegne , C. S. & Jo dan, J. A. (2002). Human pa o i us B19 binds placen al cy o ophoblas cells ia globoside ecep o . Pos e p esen ed a he IX Pa o i us Wo kshop, Bologna, I aly, 28–31 Augus 2002. Weigel-Kelley, K. A., Yode , M. C. & S i as a a, A. (2001). Recombinan human pa o i us B19 ec o s: e y h ocy e P an igen is necessa y bu no su icien o success ul ansduc ion o human hema opoie ic cells. J Vi ol 75, 4110–4116. Weigel-Kelley, K. A., Yode , M. C. & S i as a a, A. (2003). Æ51 in eg in as a cellula co ecep o o human pa o i us B19: equi emen o unc ional ac i a ion o 1 in eg in o i al en y. Blood 102, 3927–3933. Whi e, D. G., Wool , A. D., Mo ime , P. P., Cohen, B. J., Blake, D. R. & Bacon, P. A. (1985). Human pa o i us a h opa hy. Lance i, 419–421. Williams, M. D., Cohen, B. J., Beddall, A. C., Pasi, K. J., Mo ime , P. P. & Hill, F. G. H. (1990). T ansmission o human pa o i us B19 by coagula ion ac o concen a es. Vox Sang 58, 177–181. Wool , A. D. & Cohen, B. J. (1995). Pa o i us B19 and ch onic a h i is – causal o casual associa ion? Ann Rheum Dis 54, 535–536. Wool , A. D., Campion, G. V., Chishick, A., Wise, S., Cohen, B. J., Klouda, P. T., Caul, O. & Dieppe, P. A. (1989). Clinical mani es a ions o human pa o i us B19 in adul s. A ch In e n Med 149, 1153–1156. Wool , A. D., Hall, N. D., Goulding, N. J., Kan ha ia, B., Maymo, J., E ison, G. & Maddison, P. J. (1991). P edic o s o he long- e m ou come o ea ly syno i is: a 5-yea ollow-up s udy. B J Rheuma ol 30, 251–254. W igh , C., Hinchli e, S. A. & Taylo , C. (1996). Fe al pa hology in in au e ine dea h due o pa o i us B19 in ec ion. B J Obs e Gynaecol 103, 133–136. Yaegashi, N. (2000). Pa hogenesis o nonimmune hyd ops e alis caused by in au e ine B19 in ec ion. Tohoku J Exp Med 190, 65–82. Yaegashi, N., Shi aishi, H., Tada, K., Yajima, A. & Sugamu a, K. (1989). Enzyme-linked immunoso ben assay o IgG and IgM an ibodies agains human pa o i us B19: use o monoclonal an ibodies and i al an igen p opaga ed in i o.J Vi ol Me hods 26, 171–181. Yaegashi, N., Okamu a, K., Yajima, A., Mu ai, C. & Sugamu a, K. (1994). The equency o human pa o i us B19 in ec ion in non- immune hyd ops e alis. J Pe ina Med 22, 159–163. Yaegashi, N., Niinuma, T., Chisaka, H. & 7 o he au ho s (1999). Se ologic s udy o human pa o i us B19 in ec ion in p egnancy in Japan. J In ec 38, 30–35. Yo o, Y., Kudoh, T., Haseyama, K., Suzuki, N., Oda, T., Ka oh, T., Takahashi, T., Sekiguchi, S. & Chiba, S. (1995). Incidence o human pa o i us B19 DNA de ec ion in blood dono s. B J Haema ol 91, 1017–1018. Yo o, Y., Kudoh, T., Haseyama, K., Suzuki, N. & Chiba, S. (1996). Human pa o i us B19 in ec ion associa ed wi h acu e hepa i is. Lance 347, 868–869. Young, N. S. (1996). Pa o i uses. In Fields Vi ology, 3 d edn, pp. 2199– 2220. Edi ed by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippinco -Ra en. Au ho que y: Fig. 2. no ci ed in ex . A. Co co an and S. Doyle 16 Jou nal o Medical Mic obiology 53 Fig. 2. Schema ic depic ion o T- and B-cell esponse o pa o i us B19 in ec ion. Upon B19 in ec ion, B cells di ide o p oduce plasma cells and memo y cells. Plasma cells sec e e IgM an ibodies ha a e speci ic o bo h con o ma ional (N) and linea (D) epi opes o B19, which a e de ec able app oxima ely 7 days pos -in ec ion. B19-speci ic IgG is de ec able abou 15 days pos - in ec ion and is di ec ed ini ially agains bo h linea and con o ma ional epi opes o he capsid p o eins (VP1 and VP2) and, o a lesse ex en , agains NS1, bu declines agains linea epi opes o he p o eins in a ime-dependen manne . In ec ion by B19 mos likely con e s li elong p o ec ion on he hos , due o he de elopmen o memo y B cells ha a e speci ic o con o ma ional egions o he B19 capsid and also linea egions o he VP1 p o ein (Co co an e al. , 2004). An igen-p esen ing cells (APC) p ocess he i us and display B19 pep ides on hei su ace o Th cells. These Th cells hen sec e e cy okines ha play a ole in media ing an i- i us immuni y and may also be associa ed wi h pa hogenesis o B19 in ec ion (e.g. IL6 is associa ed wi h RA). Pa o i us B19 e iew h p://jmm.sgmjou nals.o g 17 Re iew O p in O de Fo m JMM Jou nal o Medical Mic obiology PAPER Please quo e his numbe in any co espondence Au ho s Da e I would like 100 ee o p in s, plus addi ional o p in s, gi ing a o al o o p in s Dispa ch add ess o o p in s (BLOCK CAPITALS please) Please comple e his o m e en i you do no wan ex a o p in s. Do no delay e u ning you p oo s by wai ing o a pu chase o de o you o p in s: he o p in o de o m can be sen sepa a ely. Please pay by c edi ca d o cheque wi h you o de i possible. Al e na i ely, we can in oice you. All emi ances should be made payable o ‘Socie y o Gene al Mic obiology’ and c ossed ‘A/C Payee only’. Tick one Cha ge my c edi ca d accoun (gi e ca d de ails below) I enclose a cheque/d a payable o Socie y o Gene al Mic obiology Pu chase o de enclosed Re u n his o m o: JMM Edi o ial O ice, Ma lbo ough House, Basings oke Road, Spence s Wood, Reading RG7 1AG, UK. 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