JOURNAL OFVIROLOGY, June 1989,p. 2422-2426 0022-538X/89/062422-05$02.00/0
Copyright© 1989, American Society for Microbiology
Propagation of Human Parvovirus B19 in Primary Culture of
Erythroid Lineage Cells Derived from Fetal Liver
NOBUO YAEGASHI,1HIROYUKI SHIRAISHI,' TOSHIKAZU TAKESHITA,' MASATAKA NAKAMURA,' AKIRA YAJIMA,3 ANDKAZUO SUGAMURAl*
Departments ofBacteriology' and Obstetrics and Gynecology,3 Tohokiu University School of Medicine, 2-1 Seiryo-machi,
and Miyagi Prefectural Instituite ofPublicHealth, 4-7-2 Saiwaicho,2 Sendai980, Japan
Received 1 December 1988/Accepted 13 February 1989
Erythroid lineage cells derived fromfetal liverweredemonstrated tobe target cells for human parvovirus B19 infection. B19 virus antigen-positive serum was inoculated into primary cultures containing erythroid lineage cells enriched fromfetal liver. The B19 virusantigenwasdetectedonabout 5% ofcellsintheculture
by immunofluorescence staining, and the stained cells were identified as erythroid lineage cells by double
stainingwithanti-B19 virus-positiveserumandanti-erythroid lineage monoclonalantibody. The immunoflu-orescencestaining study also revealedthatthe B19 virusantigenlocalized in thenucleus and theperipheryof cytoplasm. We also detectedB19virusDNA, whichwasgeneratedby replicationintheinfectedcells,notonly in thecells but alsointhe culturesupernatants, inwhich theamountof B19 DNA increaseddependingonthe period of culture, indicatingthat the cellsinfectedwith B19 virusproducedB19virus andreleased it into the medium. The ability of B19 virus released into the medium to infect fetal erythroid lineage cells was
demonstratedquantitatively. Because of the absence ofanycytopathiceffectofB19virusduringcultureperiods ofat least 15 days, this culture system should be useful in the study of B19 virus replication and in vitro generation of B19 virus. In addition, the present study may contribute to a better understanding ofthe pathogenesisofhydrops fetalis,which isprobablyassociated with B19 virus infectionduring pregnancy.
HumanparvovirusB19wasfirst discovered in theplasma of normalbloodbyCossartet al. (5). It is nowknowntobe etiologically related to human illnesses including erythema infectiosum (fifth disease) in children (2, 14), the aplastic crisis ofhemolyticdiseases(13),acuteinflammatoryarthritis in adults (12, 16), rheumatoid arthritis (4), and hydrops fetalis (1, 3, 17). Recently, several types of B19 virus have been identified genetically, but no relationship between these and the above-mentioned diseases has beennoted (7). Only erythroid progenitorcells derivedfrom bonemarrow havebeen demonstratedtobe atarget cellpopulationfor in vitro B19 virus infection (9, 10, 15, 20). When B19 viruswas inoculated into a culture of bone marrow cells, erythroid
colony formation was significantly inhibited (8). This
sug-gests that the aplastic crisis of hemolytic diseases could resultfrominfection of erythroid progenitor cells with B19 virus. However, the pathogenesis of other diseases associ-ated with B19 virus infection is still unknown. Hydrops fetalisisthoughttobecaused by infection offetalerythroid progenitorcellswith B19 virus duringpregnancy. We exam-ined whether B19 virus infected erythroid lineage cells derived from fetal liver, in which erythropoiesis occurs
actively. Ourresults demonstratethat B19virus propagates in erythroid lineage cells from fetal liver and suggest that hydrops fetalis with B19 virus infection duringpregnancy is a direct result of B19 virus infection of erythroid lineage cells.
MATERIALSANDMETHODS
Virusand antibody. At the initial stageof this study, B19 viruswasprepared from human serumfromanormalblood
donor which reacted to human anti-B19 virus-positive sera
*Corresponding author.
by counterimmunoelectrophoresis. The B19 virus antigen-positiveserum(290-6258)contained 20,ugof B19 virusDNA
perml, whichwasdetermined withaB19 virusDNAprobe, pGEM-1/B19 (the generous gift of J. P. Clewley). Human anti-B19virus-positive serawerealso obtained fromnormal blood donors.
EP-1 monoclonal antibody (immunoglobulin M [IgM]) specificfor the surfaceantigenoferythroidlineagecells(19)
was used in double immunofluorescence staining experi-ments.
Preparationof fetalerythroidlineage cells.Liver tissuewas
obtained from aborted fetuses ranging in gestational age
from 18 to 21 weeks. Theparents agreed to theuse ofthe tissue for research studies. These tissueswerewashedthree times in
Ca2"-free
Hanks medium, dissected into small pieces, and treated twice with collagenase (Wako Co., Osaka, Japan) at 37°C for 15 min. Hepatocytes were then removed by centrifugationat50 x gfor 1 min. Thesuper-natant was further centrifuged on a Ficoll-Conray gradient
(specific gravity,1.077g/cm3),and cellsatthe interfacewere
collected. Cells were cultured at 5 x 106/ml in RPMI 1640 medium supplemented with 20% fetal calf serum (FCS),
antibiotics, and 20 U of recombinant human interleukin-3 (IL-3; obtained from the Genetics Institute, Boston, Mass.)
perml at37°C inahumidified atmosphere with 5% CO2for 16 h in plastic culture flasks (Corning Glass Works, no.
25160-225). Subsequently, nonadherent cells were recov-ered. Over 70% of the cells were found to be erythroid lineage cells by Wright Giemsa staining, and about 20% of them were stained with EP-1 monoclonal antibody. These wereused asthetargetcellsfor the B19 virus infection.
Virus inoculation and cell culture. Cells(108) ofan eryth-roid lineage-enriched fraction were incubated with 20
RI
of B19 virusantigen-positive serumfor 2 hat4°Ctoadsorb the2422
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virus. The cells were washed three times with RPMI 1640 medium, adjusted to 5 x 106 cells per ml with RPMI 1640
medium containing 20% FCS, IL-3 (100 U/ml), and 1 U of
sheep erythropoietin (Connaught Laboratories, Ontario, Canada) per ml and cultured at 37°C in a humidified atmo-sphere with 5% CO2. Mock infection was carried out in a
similarmanner with B19virus antigen-negative serum. Cells
and supernatants were harvested separately from day 0 to day 15.
Immunofluorescence staining. B19 virus-infected cells were washed twice with phosphate-buffered saline (PBS) and mounted on glass slides. Cells were fixed in
acetone-methanol (1:1) at -20°C for 20 min. Preparations were
treated with10 p.1 of a 1:20 dilution of anti-B19virus-positive
serum for 30 min at 37°C, washed twice with PBS, and
stainedwith 10 p.1of a 1:50 dilution of fluorescein
isothiocy-anate (FITC)-conjugated goat anti-human IgG for 30 minat 370C.
Indoubleimmunofluorescence staining, 5 x 106cells were treated with 20p.lof a1:100 dilution of EP-1 ascites fluid for 30 min at 40C. The cells were washed twice with PBS
containing 0.5% bovine serum albumin and then incubated
with 20
p.l
of a 1:20 dilution of FITC-conjugated goatanti-mouseIgMfor 30 min at4°C.After being washed twice
with PBS, the cells were fixed and stained with anti-B19 virus-positive serum and rhodamine-conjugated goat
anti-human IgG.
Dotblothybridization. B19virus-infectedcells werelysed in 0.01 MTris hydrochloride buffer(pH 7.5) containing0.5%
sodium dodecyl sulfate (SDS), 0.1 M NaCl, and 0.005 M
EDTA, and the lysate was homogenized with a taper-type apparatus. DNA was extracted from the homogenized ly-sate, culture supernatants, and B19 virus antigen-positive serum by incubation with 200 p.g ofproteinase K per ml
overnight at 370C and then by treatment with phenol and
chloroform. DNA wassubjectedtodot blothybridizationby
amodifiedmethoddescribedpreviously (10). In brief, 0.6 N
NaOHwasaddedto DNAsamples fordegeneratingRNAs.
Theywereincubatedat60°C for30 minandthenneutralized
with 2 Msodium acetate. DNA (5
p.g)
from the cellextracts and DNAfrom50 p.1ofthe culture supernatants were diluted with lOx SSC (lx SSC consists of 0.15 M NaCl, 0.015 Mtrisodium citrate, pH 7.0) and applied to nitrocellulose
membranes. Afterbaking at 80°C for3 h, membranes were
prehybridized with 2 mg of denatured salmon sperm DNA
and 2mgofdenatured tRNA in ablotting solutionconsisting
of50% formamide, 10%dextran sulfate, 0.1% SDS,0.05 M
Tris hydrochloride (pH 7.5), 5x Denhardt solution (1x is
0.1%Ficoll[PharmaciaFineChemicals], 0.1% polyvinylpyr-rolidone, 0.1%bovineserum albumin), and Sx SSC at 42°C
overnight. DNA on the membranes was hybridized with a
B19 virus-specificDNA probe, the5.2-kilobase-pairEcoRI
fragment ofpGEM-1/B19, labeledwith
32p
via nicktransla-tion, in thesolution described above at42°C overnight.
Preparation of B19 virus from in vitro culture. A 2-ml amount of the supernatant from the B19 virus-infected cell
culture was layered on 2 ml ofa 30% sucrose cushion and
centrifuged at 35,000 rpm witha Hitachi RPS-56Trotorfor
15h at 4°C. The B19 virus pellet was suspended in 20 pL. of PBS. The resultant B19 virus was tested for its ability to infecterythroid lineagecells in vitro.
RESULTS
Infection of erythroid lineage cells from fetal liver. We examined the infectivity of B19 virus for
erythroid
lineage3.0
z 2.0
0,l
m 1.00 r h
0 1 2 4 8 15
Days post-infection
FIG. 1. AppearanceofB19virus-infectedcells in the fetal eryth-roidlineage cell culture. Erythroid lineage cells derived fromfetal liver wereincubated withB19virus antigen-positive(@)or-negative (0) serum. They were then cultured for 15 days, and B19 virus-infectedcells weredetected by indirect immunofluorescence stain-ing.
cells from fetal liver. For this experiment, fetal liver cells were fractionated, and an erythroid lineage cell-enriched fraction was obtained in which more than 70% of the cells were of erythroid lineage, as determined by cytological
examination. This fraction was cultured in the presence of
B19 virus antigen-positive serum for 15 days. Cells (106) were harvested every day, and the expression of B19 virus
antigenwasexaminedby indirect immunofluorescence
stain-ing.Approximately 1.0%of the cells were stained onday 1,
but the fluorescence intensity was much weaker than that
observed on later days. On day 4, the intensity of
fluores-cence in the positivecells becamemaximal and the percent-age increased to 5.0%. After day 8, the numberofpositive
cells decreased gradually (Fig. 1). B19 virus-positive cells
variedfrom 1 to5%withthepreparation oftargetcellsfrom
liver. With B19 virusantigen-negative serum, nobrightcells were found after immunofluorescence staining. Similar re-sults were obtainedwith B19viruspurified fromserum(data not shown).
To confirm the specific infection with B19 virus, we
carried out an experiment to test the ability of human anti-B19 virus-positive sera to neutralize B19 virus.
Anti-B19virus-positive serum(20
p.l)
wasmixed with 2p.l of B19virus antigen-positive serum. The mixture was left to stand for 30 minatroomtemperature and thenwasinoculatedinto the culture. Infection with B19 virus in the cells was com-pletely neutralized bytheaddition of anti-B19virus-positive serum but notbynegative serum (Fig. 2). The immunofluo-rescence staining ofB19 virus-infected cells indicated that B19 virusantigen localized in the nuclei and peripheries of
cytoplasms(Fig. 3).
Although erythroid lineagecells in the fraction used were
enriched by the
procedure
describedabove,
itobviously
containedothercelllineage
populations
aswell. We wishedto identify the lineage of cells that were infected with B19
virus. Cells from day4 of culture inoculated withB19 virus were stained doubly with human anti-B19
virus-positive
serum and mouse monoclonal antibody EP-1, which is
specific for burst-forming
unit-erythroid cells,
colony-forming
unit-erythroid
cells,
anderythroblasts (19).
Cellsstained with anti-B19
virus-positive
serum werealways
stained with EP-1, and about 20% of
EP-1-positive
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[image:2.612.346.512.74.243.2]2424 YAEGASHI ET AL.
~, 1.0
0
co0)
0 4 8
Days post-infection
FIG. 2. Neutralization of B19 virus by human antisera. B19virus antigen-positive serum was incubated with humananti-B19 virus-positive (serumA[0]andserumB [A]) or-negative (serum C[0] and serumD[A])sera for 30min at roomtemperature. These four serumpreparations were then inoculated intotheprimary culture of cryopreserved fetal erythroid lineage cells, andB19virus-infected cellsweredetectedby indirect immunofluorescence staining. expressed B19 virusantigen (Fig. 4). These results indicate that B19 virus can infect erythroid lineage cells in human
fetal liver and that the expression and replication of B19
virus can occur in the cells.
Replication of B19 virus genome in the infected cells.
Replication ofviral DNA in the erythroid lineage cells was
examined byquantitatively determiningtheamountof viral
DNAinthecellsandculture supernatants. Weusedthedot
blot hybridization method and estimated the concentration
of viral DNA in samples by using cloned whole B19 virus
DNAfrom pGEM-1/B19 as astandard. As littleas 10 pgof
B19 virus DNA per spot could be detected in this system
(Fig. 5a, lane P). Cells fractionated from fetal liver were
washed three times after exposure to B19 virus
antigen-positive serum. The medium solution used for the third
washing was tested for detection of B19 virus DNA. No
positive signal for B19 virus DNA was found in the assay
system (Fig.
5b,
day0). DNAwasextractedfrom cells and culture supernatants separately harvested from the B19virus-infected cell culture on the days indicated in Fig. 1.
DNA
(5
jg)
from the cells or DNA from 50 ,lI ofculture supernatants wasspottedas asampleof 1x onto nitrocellu-lose membranes. B19virus-specificDNAcould be detected in the sample on day 1 postinfection and was maximally presentin the sample from cells on day 2, atwhich time the amountof B19 virusDNA wasestimatedtobe5 ngin the1xspot containing 5 ,ug of whole DNA, equivalent to the
amountof DNAfrom 2 x 106cells. As shown in Fig. 1, 5% of the cells included the B19 virus genome. Thus, we estimated the number of B19 viruses in an infected cell to be about 2 x 104. Even a DNA samplefrom the day 15 culture showed the existence of the B19 virus genome in the cells
(Fig. 5a). B19 virus-specific DNA was similarly detected in
preparations from culture supernatants. The content of B19 virus DNA gradually increased from 0.2 ng in 1 ml of culture supernatant onday 1 to 20 ng/ml on day 15 (Fig. Sb). These results indicate that the B19 virus genome replicates in
erythroid lineage cells from human fetal liver and that B19
virionsarereleased from these cells.
Infectivity of B19 virus produced in vitro. We next
ad-FIG. 3. Immunofluorescencestainingof B19virus-infected cells. Fetal erythroid lineage cells were infected with B19 virus (A) or mock-infected(B)in a manner similar to thatdescribedinthelegend toFig. 1.Onday4postinfection,thecellswerestainedby indirect immunofluorescence. Original magnification, x1,680.
dressed the question ofwhether the B19virus
generated
in the invitro systemwasinfectious.B19virus in the superna-tantfrom the culture 15days after infection washarvestedandconcentrated bycentrifugation. Threedoses of the B19
virus preparations were inoculated into erythroid lineage
cell-enriched fractions from fetal liver, and the cells were cultured. Cellswereharvestedondays4and 8 afterinfection and tested for the expression of B19 virus by indirect immunofluorescencestainingand dot blothybridization.B19 virus-positive cells appearedonday 4, and the percentageof
positive cells was dependent on the dose of B19 virus
inoculated (Fig. 6).
B19virus-specific DNA wasalso detected in cellextracts
of the culture from days 4 and 8. The amount of B19
virus-specificDNAdetected was afunction of theamountof
B19 virusinoculatedinitially (Fig.7). Basedontheintensity
of signals
hybridized
with the B19 virus probe, the copynumber of the B19 virus genome inaninfected cellonday4
wasestimated tobe about3 x 104. Although the amountof
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0.9
FIG. 4. Double immunofluorescence staining. Fetal erythroid lineage cells infected with B19 virus were harvested on day 4 postinfection and stainedsequentially with EP-1 (A) andanti-B19 virus-positive serum (B) as described in Materials and Methods. Original magnification, x1,630.
B19virusproducedby this culture system was much smaller
than that with B19 virus from serum, the infectivity per
microgramof DNA did notdiffer significantly between B19
virusproduced in vitro and in vivo (Fig. 5 and 7).
(a)
Days post-infection2
51)
a)
.I-0)
a)
0m
0.8
0.2
0 4 8
Days post-infection
FIG. 6. Infectivity ofB19virusproduced by thefetalerythroid lineage cell culture.The supernatant ofthe B19virus-infected fetal erythroid lineage cell culturewasharvestedonday15postinfection and serially concentrated 4 (D), 20 (A), and 100 times (0). The concentratedsupernatantswereinoculatedinto theprimaryculture ofcryopreserved fetalerythroid lineage cells,and B19virus-infected cells were detected by indirect immunofluorescence staining. B19 virus antigen-positiveserum(0)wasinoculatedin asimilarmanner as acontrol.
DISCUSSION
Asthe serumofpatientswith B19 viremiaisknown as the onlysourceofB19virus,establishmentofanin vitrosystem
to provideB19 virus stably is urgentlyrequired. B19 virus
has been demonstrated to replicate in erythroid progenitor
cellsderived from thebone marrow ofpatientswith
hemo-lytic anemias (9, 10) and from normal human bone marrow
(15).
0 1 2 4 8 15 S P
Ix
l
lox a ox
booox
(b)
c 1X
-_ lox
1 ioox
**.@ 0.
* 0 .0
* ~~00
Days post-infection
0 1 2 4 8 15 S P
*...0 0.0
[image:4.612.54.291.75.346.2]0*
FIG. 5. Viral DNAproduction in the B19virus-inoculated eryth-roidlineage cell culture. DNAwasextracted from the cells (a) or
culture supernatants (b) harvested in the experiment described in thelegendtoFig. 1. Their B19 virus DNAcontentsweredetermined
by dot blot hybridization with a B19 virus DNA probe,
pGEM-1/B19. DNAextractedfrom 1 of B19 virusantigen-positiveserum
(S)and10ngof DNAof thepGEM-1/B19 probe (P)wereusedas
controls. TheseDNAsampleswereserially diluted with 1Ox SSC.
Days post-infection
0 4 8
..
.
IA B C A B C
0
Ix lox 10ox 1oOox
s P
0 *0 @0
* S
FIG. 7. Detection of viral DNA in fetal erythroid lineage cells infectedwith invitro-propagatedB19virus. Fetalerythroidlineage cellsinfectedwith B19viruspropagatedinvitro wereharvestedon
days 0, 4, and 8 postinfection in the experiment described in the legendtoFig.6.Dotblothybridizationwith thepGEM-1/B19 probe wasperformedon5-,ug DNAsamplesprepared fromtheerythroid lineage cells infected with B19 virus preparations, which were
culture supernatantsconcentrated4(A), 20(B),and 100 times(C). DNAextracted from 1 ,ulof B19 virus antigen-positive serum (S) and 10ng of DNA ofpGEM-1/B19(P)wereusedascontrols. These DNAsampleswere serially dilutedwith lOx SSC.
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Inthe present study, wealso attempted to developan in
vitro culturesystemforB19 virusproductionwithfetal liver.
We demonstrated that B19 virus was susceptible to and
replicated
in erythroid lineagecells derived from fetal liverby
doubleimmunofluorescence stainingwithanti-B19virus-positive serum and monoclonal antibody EP-1, specific for
erythroid lineage
cells. B19 virus infection of the livererythroid
lineage
cells was similar to that of bone marrowerythroidprogenitorcells with regardtoviral DNA
produc-tion(10).B19virusDNAassociatedwith the cellsreacheda
maximum levelfrom 2 to 4 days afterinfection, while B19
virus DNA in the culture supernatants increased gradually
with timeinboth liverandbonemarrowsystems. In theliver
erythroid
lineage
cell system, the B19 virus DNAcontentinthe supernatant reached 20
ng/ml
on day 15 postinfection.Thiswastwiceasmuchasin the bonemarrow system(10).
B19virusantigenwasdetectedondays 1to 15in theliver
erythroid
lineage cells,
but onlyondays
1 and 2in the bonemarrowerythroid progenitor cells (10, 20). Theantigen was
localizedinnucleiand the
peripheries
ofthecytoplasmin theliver erythroid lineage cells. A similar feature of antigen
expression
has been observed in cells infected with otherparvoviruses,
such asadeno-associated virus(6)andbovineparvovirus (11). In the bone marrow erythroid progenitor
cells,
however,
B19 virusantigenwasdetectedmainly
in thecytoplasm,
not in nuclei (10, 20). Thisdiscrepancy
in B19virus antigen localization between liver and bone marrow
erythroid lineage
cells isprobablyduetothedifferencein the culture systems. Livererythroidlineage
cellswerecultured in the presence of IL-3, known as a pluripotent stem cellgrowth factor (18), which may allow
erythroid
progenitorcellsto survive
longer
andto accumulate B19 virusantigen
innuclei. Thisexplanation isalsosupported bythefactthat
hardly
anycytopathic
effect in the livererythroid
lineage
cells infected withB19 virus wasobserved. The percentage
of B19 virus-infected cells was
higher
in the bone marrowerythroid
progenitor
cells than in the livererythroidlineage
cells (10), but the recoveryofB19virus in culture superna-tants was the opposite, as mentioned above. This may be
also due to the difference in the survival of cells between
these two culture systems.
There have been several reportssuggestingthat B19virus
infection
during
pregnancy causeshydrops
fetalis(1, 3, 17).B19virus infectionin thefetushas beenspeculatedtocause
the destruction oferythroidprogenitor cells, generatingthe
development
of severe anemia and heart failure.Conse-quently, the fetus falls into a hydropic state, resulting in
intrauterine fetaldeath.Our presentstudydemonstrates that
fetal
erythroid lineage
cells recovered from the liver are targetcellsforB19virusinfection,supportingthenotion that B19virus infection inducespathogenesis of hydropsfetalis.Finally, although
fetal liver erythroid lineage cells arevaluable for in vitro propagation of B19 virus, a stable cell
line permissive for B19virus infection would be preferable.
Because of the excellent cell viability, the present culture systemof fetal livererythroid lineage cells may be useful for
establishing
a stable target cell line by the hybridoma method.ACKNOWLEDGMENTS
WethankJ. P.ClewleyforthegenerousgiftofpGEM-1/B19and T.Yokochi forhis kindgift ofEP-1.
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