Vol.66, No. 2 JOURNALOFVIROLOGY, Feb. 1992, p. 1171-1175
Copyright C) 1992, American Society for Microbiology
Pathogenesis of Classical
Deficiency Caused by
SUSA,ltMATTHIAS KONIG,1 ARMIN SAALMULLER,1 MATTHIAS J. REDDEHASE,2 ANDHEINZ-JURGEN
Federal Research Centrefor Virus Diseases of Animals, D-7400 Tubingen,' andDepartmentof Virology,
University of Ulm, D-7900 Ulm,2 Germany
Received 6 June 1991/Accepted4October1991
Hogcholera, also knownasclassicalorEuropean swine fever,is caused by hog cholera virus,amemberof thegenusPestivirus. It is shown here that the endstageof lethal infectionin the natural host is associated with
adramatic depletion preferentiallyof B lymphocytes in the circulatorysystemaswellasin lymphoid tissues.
Alreadyattheonsetof disease, viral replication in lymphoid tissuesdemarcates the germinalcenters,and the
viralgenomeremains localizedtothatsiteasthe diseaseprogresses evenafter morphologic disintegrationof the
follicularstructure. A blockin B-lymphocyte maturation by infection and destruction of germinalcenters is
discussedas akeyeventinthe pathogenesis ofacute,lethal hog cholera.
Earlystages in acute hog cholera are associated with fever
diarrhea.The progression of the disease is accompanied
byaseverewasting syndrome. The terminal stage is signified
discoloration oftheskin and weakness of the hind
legs and is sometimesaccompanied by neurological
Disseminatedintravascular coagulopathy, extensive
tissue hemorrhages,and thymus atrophy are seen at autopsy
reference20). A virus with a
of 12,284 nucleotides(13)
of hog cholera(11)
is accordingly referredto as
virus(HCV). Together with the structurally and
serologically related bovine viral diarrhea virus ofcattle (6,
border disease virus of sheep,HCV is grouped
into thegenus Pestivirus (23).
basis ofrecent studies
translationand the genome
bovine viral diarrheavirus (4,
5)aswell as HCV (13, 19), the
currently valid taxonomic classification ofPestivirus as a
family Togaviridae(23) has been
Immunological implications of HCV infection, including
disease, have been described
previously (for reviews,see
insights into the unique organization ofthe
2, 12, and 14) andthe
of the viralgenome
(13)that made it
reinvestigate the effect of HCVon
of conclusions drawn
viral destruction of
tissues is themost
conse-quence of acute HCV
Three-month-old outbred GermanLandrace swine were
50% tissueculture infective
doses of HCV strain Alfort. Disease
tPresentaddress: Department of
animals died betweendays 10 and 20
after infection,and the
moribundstage. At the terminal
stage,virus titers in sera ranged
between3.6 and 6.1 log50%
occasionally detectable (datanot
documented by differentialblood cell counts by
Pappenheim stain (Fig.1).
Notably, band form
neutrophils increased in absolute number, while other blood
Owing to the variance in the progression of disease,
S 0630 S 0631 S 0528
0 9 16 0 9 18 0 5 9
days post infection
FIG. 1. General leukopenia during acute hog cholera. Blood samples were taken from the venae cavae externae of individual swine before infection (day 0) and during the course of disease induced by intranasal infection with 7
log50% tissue culture infective doses of HCV strain Alfort. Differential
leukocytecounts weremadebyusing thecoverslipmethodof blood film
followed byPappenheim staining.Abbreviations: Mo, monocytes;
BN, band formneutrophils; SN, segmented
on November 10, 2019 by guest
1 2 3 4
S0630 4 0 39 16
S0629 31 1 47 19
S0312 14 1 49 25
Medbn 27,51 48 20
1 2 3 4
S0630 28 3 47 20
S0629 46 2 31 17
S0312 U 4 41 S
1 2 3 4
S0630 50 3 31 6
S0629 78 2 12 5
S0312 63 2 32 2
Medin 64.5 2 30 4.5
FIG. 2. Relativechanges in leukocytesubpopulations.Cytofluorometric
(top)and at twodifferent stages of disease(centerandbottom). Dextran(Ficoll
antibody 8/1 and allophycocyanin-conjugated streptavidin. The measurements were
performedon a dual-laser FACStar Plus
Dickinson, MountainView, Calif.). Inthetwo-dimensional contourplotsof 8/1antigenexpressionversus
SSC,frames indicateelectronic windows set ongranulocytes (window 1), monocytes(window 2),T
lymphocytes(window 4).Contour lines representlevels of 5, 10, 20, 40, 60, 80, 100, 120, 140, and 160cells,withatotal of25,000cellsanalyzed.Datain the tablesarethe percentages of cells located in therespectivewindows, compiled from data for four individual swine,with thedepictedcaseunderlined.
on November 10, 2019 by guest
VOL. 66, 1992
n- S Median -24%
n-S Median= 6 %
n-3 Median -38%
n =2 Median= 16%
FIG. 3. Depletion of B lymphocytes in peripheral blood and lymphoidtissues. Ficoll-Hypaque-enriched mononuclear leukocytes from peripheral blood, spleen, and mandibularlymph nodeswerestained forcytofluorometricanalyses with monoclonal antibody 1G6, specific for
theporcine Ig kappa chain, and fluoresceinisothiocyanate-conjugated F(ab')2fragments ofgoatanti-mouseIgG-F(ab')2. Foreachanimal, 25,000 cellswereanalyzed throughout. Thevertical marker delimits the negative control defined by staining with the fluorescentconjugate intheabsence of the specific first antibody.The numbers in theupperleft and rightcorners arethepercentagesof Ig-negativeand Ig-positive cells, respectively.
relative changes in peripheral blood leukocyte
subpopula-tions were not monitored at fixed time points but were
instead related to the onset of disease, indicated by fever,
which usually occurred around day 5, andtotheendstageof
disease, whichwassignified byhemorrhages of the skin and
a fall inbody temperature to below normal. Fora random
sample of four swine, analyzed individually before infection
andduring thecourseofdisease, resultsarecompiled in Fig.
2. The porcine leukocyte antigen 8/1 is known to be
ex-pressed by all porcine leukocytes with the exception of those
ofB-lymphocyte lineage (10, 17, 18). In acytofluorometric
analysis, detection of this antigen together with the side
scatter(SSC), aphysical parameterreflecting cell
granular-ity, allows for a single-step quantitation of the four major
leukocyte subpopulations. In the two-dimensional contour
plot of 8/1 antigen expressionversus SSC (Fig. 2),
granulo-cytes are described as 8/12+SSChi (electronic window 1 in
Fig. 2), monocytesaredescribedas
resting T lymphocytes aredescribed as
3), and B lymphocytes aredescribed as
4). It is evident fromthe data that theprogressionof disease
isassociated witha significantincrease in theproportion of
granulocytes andwithanalmostcompleteloss of B
lympho-cytes, whereas the reduction in T lymphocytes is less
impressive. In absolute terms, the number ofgranulocytes
also decreases. However, the augmentation of band form
neutrophilsoversegmented neutrophils (Fig. 1) indicatesan
increasedrateof granulocyte production inresponsetoHCV
Alterations within the T-lymphocyte compartment and
their significance in disease are the subjects of current
research, but it is informative thatamong thefour
subpop-ulations ofTlymphocytes knowntobepresentin swine(16),
CD4- CD8+ T lymphocytes relatively increase in number,
number and thesubpopulationsCD4+CD8+ and
CD4+CD8-areessentially unchanged in peripheral blood (unpublished
One could raise the objection that the scarcity of B
lymphocytesin peripheral blood may result from their
re-cruitmentto lymphoid tissue rather than indicate their
de-pletion. We thereforeextended thecytofluorometric analysis
to lymphoid tissue, represented bythe spleen and by
man-dibular as well asinguinal lymph nodes. Mononuclear
leu-kocytes wereenriched, and B lymphocyteswere then
posi-tively identified by staining of surface immunoglobulin (Ig)
(Fig. 3). The results substantiate the conclusion that B
lymphocytes are indeed depleted at the terminal stage of
disease. B lymphopenia developed independently of the
routeofinfection, intranasalorintramuscular, andwasalso Lymph Node
al !i ..
on November 10, 2019 by guest
IF.,A 4' o
FIG. 4. Localization of HCV genome ingerminalcentersinlymphnodes.
Immunocytochemistry detectingcellsthat express
(Aand C) and in situhybridization detectingHCV RNA(B andD) insections of mandibular
disease,in this case atday5after intranasal infection (A andB),andatday16inthe end stage of disease
sections of frozen tissuewerefixed inacetoneand labeled with monoclonal
biotinylated anti-mouse serum andanavidin-biotin complex
Calif.). Insituhybridizationwasdone withproteinase K-treated,
of35S-labeledribonucleotideprobesrepresenting mostof the viral RNA. Allsectionswerecounterstained with
hematoxylin-eosin (B and D) andareshownatthesamemagnification. Bar, 100,m.
in swine infectedwith the more
HCV, whichcauses death between 6 and 10
final linkin the
detection of the HCVgenome
exemplified in Fig.4
for mandibular lymph
epithelialcells of the
tonsils,as well as in B
nodes,and the Peyer's
infection ofendothelial and other
cells occurs later (lla). It should be remembered that, in
swine, lymphnode tissue is microanatomically inverted in
homingareas of B and Tlymphocytes are interchanged
lymphocyteslocated in the lymph node cortex and B
follicles in the medulla (la). Immunocytochemistrywith
lymphocytesshows an intact
disease (Fig.4A) and remnants
beennoted in earlier
descriptions of hogcholera
tissues,acausative role in the
be evaluated and the
attrib-uted to a
of the virus.We therefore
findingthatviral RNA localizes
specificallytoBfollicles at a very
(Fig. 4B),when the
is still intact
ribonucleotides transcribed fromseveral
cDNA clones used as a
secondary follicle while
location ofthe HCV genome
follicle isthe site
unequivocallydemonstrates that it is a
microanatomicalsite of virus
on November 10, 2019 by guest
cating the remnant of a follicle (Fig. 4D). Apparently, infection did not significantly spread into interfollicular lymph node tissue.
We are currently considering two alternatives for explain-ing B-lymphocyte depletion. First, B lymphocytes in any
maturation within follicles(for a review, see
refer-ence 9), that is, centroblasts, centrocytes, and B blasts,
could themselves be the cellular targets of HCV, or,
alter-natively, they could be deprived of critical cytokines
infectionof the supporting follicular dendritic
network.We plan to address the targetcell question by
tryingto detect HCV gene expression in sorted
ofB lymphocytes from infected swine.
Conclusion. The data have shown that HCV replication in B
follicles isa specific and early event in hog cholera and
precedes generalized infection. Clearly,depletion of B
lym-phocytescannot account for all the pleiotropic
itis generally held that antibodies against HCV can be protective and as recovery from acute infection is known to be associated with seroconversion (for
reviews,see references 8 and 21), it appearsjustified to us to
propose that B-follicle tropism of an HCV isolate is an
important determinantfor the course of disease.
We thank Laura Archetti and M. Amadori(Istituto Zooprofilat-tico, Brescia, Italy) for providing antibodies to porcine Ig and B. Liess (Veterinary School, Hannover, Germany) for HCV strain Alfort. F. Weiland helped establish immunocytochemistry. We appreciate the support and continuous interest of U. H. Koszi-nowski (Departmentof Virology, University of Ulm, Ulm, Germa-ny), who had originally initiated the project. The technical assis-tance of Petra Ulrich, Sabine Maurer, Silke Mauritz, and Anne
Straubinger and the secretarialhelp of Ingrid Bennettisgratefully
This work was jointly supported by the Bundesminister fur Forschung und Technologie and IntervetInternational BV(project no. 0319028A to H.-J. Thiel) and by the Deutsche Forschungsge-meinschaft (grant Re 712/1-2toM.J. Reddehase).
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