Pathogenesis of classical swine fever: B-lymphocyte deficiency caused by hog cholera virus.

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Vol.66, No. 2 JOURNALOFVIROLOGY, Feb. 1992, p. 1171-1175

0022-538X/92/021171-05$02.00/0

Copyright C) 1992, American Society for Microbiology

NOTES

Pathogenesis of Classical

Swine

Fever:

B-Lymphocyte

Deficiency Caused by

Hog

Cholera

Virus

MILORAD

SUSA,lt

MATTHIAS KONIG,1 ARMIN SAALMULLER,1 MATTHIAS J. REDDEHASE,2 ANDHEINZ-JURGEN

THIEL'*

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.

Early

stages in acute hog cholera are associated with fever

and

diarrhea.

The progression of the disease is accompanied

by

aseverewasting syndrome. The terminal stage is signified

by

a

blue

discoloration of

theskin and weakness of the hind

legs and is sometimes

accompanied by neurological

symp-toms.

Disseminated

intravascular coagulopathy, extensive

tissue hemorrhages,

and thymus atrophy are seen at autopsy

(for

a

review,

see

reference

20). A virus with a

positive-stranded

RNA

of 12,284 nucleotides

(13)

is

the

etiological

agent

of hog cholera

(11)

and

is accordingly referred

to as

hog cholera

virus

(HCV). Together with the structurally and

serologically related bovine viral diarrhea virus of

cattle (6,

7) and

the

border disease virus of sheep,

HCV is grouped

into the

genus Pestivirus (23).

On

the

basis of

recent studies

of the

strategy

of

translation

and the genome

organization of

bovine viral diarrhea

virus (4,

5)

aswell as HCV (13, 19), the

currently valid taxonomic classification of

Pestivirus as a

genus

of the

family Togaviridae

(23) has been

questioned

(3).

Immunological implications of HCV infection, including

general leukopenia

during

disease, have been described

previously (for reviews,

see

references

8

and

21).

New

insights into the unique organization of

the

immune

system

of swine

(for reviews,

see

references

2, 12, and 14) and

the

cloning

of the viral

genome

(13)

that made it

possible

to

identify

tissue sites

of viral

replication

by molecular

probes

led

us to

reinvestigate the effect of HCV

on

the immune

system. In

revision

of conclusions drawn

by earlier

investi-gators

(22)

we

document that

B-lymphocyte

deficiency

caused

by

viral destruction of

germinal

centers

in

lymphoid

tissues is the

most

significant

pathoimmunological

conse-quence of acute HCV

infection.

Three-month-old outbred German

Landrace swine were

infected

intranasally

with

7

log

50% tissue

culture infective

doses of HCV strain Alfort. Disease

leading

to severe

symptoms

developed

in all

animals tested.

Four

of

12

*

Corresponding

author.

tPresentaddress: Department of

Virology,

University

ofUlm,

D-7900Ulm, Germany.

animals died betweendays 10 and 20

after infection,

and the

others were

sacrificed

in a

moribund

stage. At the terminal

stage,virus titers in sera ranged

between

3.6 and 6.1 log50%

tissue culture

infective

doses, and

neutralizing antibodies

to

HCV

were

occasionally detectable (data

not

shown).

For

three individual

swine,

general

leukopenia

in peripheral

blood

was

documented by differential

blood cell counts by

using

the

Pappenheim stain (Fig.

1).

Notably, band form

neutrophils increased in absolute number, while other blood

cell

populations progressively

declined.

Owing to the variance in the progression of disease,

S 0630 S 0631 S 0528

15

*Ly

,110

L

80

0

"4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~E"

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

log

50% tissue culture infective doses of HCV strain Alfort. Differential

leukocyte

counts weremadebyusing thecoverslipmethodof blood film

preparation

followed byPappenheim staining.Abbreviations: Mo, monocytes;

BN, band formneutrophils; SN, segmented

neutrophils; Ly,

lym-phocytes. 1171

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1172 NOTES

0domhhin_2m

Onsetof1lsem

End-Stageof

mm-±z

0D

Swims

Window

1 2 3 4

S0630 4 0 39 16

S0631 24

4

0

21

S0629 31 1 47 19

S0312 14 1 49 25

Medbn 27,51 48 20

S_im

Window

1 2 3 4

S0630 28 3 47 20

S

0631

i

2

LX

I

S0629 46 2 31 17

S0312 U 4 41 S

Medan

36

2.5

4 17.5

1 2 3 4

S0630 50 3 31 6

S0631

6i

Q

22

i

S0629 78 2 12 5

S0312 63 2 32 2

Medin 64.5 2 30 4.5

SSC-b

FIG. 2. Relativechanges in leukocytesubpopulations.Cytofluorometric

analyses

of

peripheral

blood

leukocytes

before infection

(top)

and at twodifferent stages of disease(centerandbottom). Dextran(Ficoll

400)-enriched

leukocytes

werestained with

biotinylated

monoclonal

antibody 8/1 and allophycocyanin-conjugated streptavidin. The measurements were

performed

on a dual-laser FACStar Plus

(Becton

Dickinson, MountainView, Calif.). Inthetwo-dimensional contourplotsof 8/1antigenexpressionversus

SSC,

frames indicateelectronic windows set ongranulocytes (window 1), monocytes(window 2),T

lymphocytes

(window3),and B

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.

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VOL. 66, 1992

PeripheralBlood

Before Infection

Spleen

71

29

fluorescence,.n.e...-nluorescenlceintensity

n- S Median -24%

n-S Median= 6 %

n-3 Median -38%

n =2 Median= 16%

End- Stageof

Disease

90

10

fluorescenceintensity 0

10.

I1g 0

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

8/13+SSC'°

(window 2),

resting T lymphocytes aredescribed as

8/1+SSC'°

(window

3), and B lymphocytes aredescribed as

8/1-SSC'°

(window

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

infection.

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,

whileCD4- CD8-

y/8

Tlymphocytes(14)arediminished in

number and thesubpopulationsCD4+CD8+ and

CD4+CD8-areessentially unchanged in peripheral blood (unpublished

data).

One could raise the objection that the scarcity of B

lymphocytes

in 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

Median-21 %

Median- 7%

n-3

n-2

89

11

al !i ..

3...2...

A4

NOTES 1173

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1174 NOTES

,=t,

t~~~~~~~~.

T,- s

...'....,'

N0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~w

OP

IF.,A 4' o

~~J0'~

o!:<:.~~~~~~~~~~~~~~~~~~~~~F.

,

AL

,

,;,

,

iA

.

.

F4

~O

*0_____

FIG. 4. Localization of HCV genome ingerminalcentersinlymphnodes.

Immunocytochemistry detecting

cellsthat express

IgM

(A

and C) and in situhybridization detectingHCV RNA(B andD) insections of mandibular

lymph

nodesobtainedattheonsetof

disease,

in this case atday5after intranasal infection (A andB),andatday16inthe end stage of disease

(C

and

D).

For

immunocytochemistry,

cryostat

sections of frozen tissuewerefixed inacetoneand labeled with monoclonal

antibody

2E8toswine

IgM.

Staining

was

performed

by using

a

biotinylated anti-mouse serum andanavidin-biotin complex

immunoperoxidase

(ABC kit,

Vectastain;

Vector

Laboratories, Burlingame,

Calif.). Insituhybridizationwasdone withproteinase K-treated,

paraformaldehyde-refixed,

and

acetylated

paraffin

sectionswithamixture

of35S-labeledribonucleotideprobesrepresenting mostof the viral RNA. Allsectionswerecounterstained with

hematoxylin (A

and

C)

or

hematoxylin-eosin (B and D) andareshownatthesamemagnification. Bar, 100,m.

apparent

in swine infected

with the more

virulent

Brescia

strain of

HCV, which

causes death between 6 and 10

days

after

infection (data

not

shown).

The

final link

in the

chain

of evidence

came

from

the

detection of the HCV

genome

in

germinal

centers

of

lym-phoid

organs, as

exemplified in Fig.

4

for mandibular lymph

nodes.

It

is of

interest

that HCV

replication

is seen

first

in B

follicles and

epithelial

cells of the

tonsils,

as well as in B

follicles of

the

regional

lymph

nodes,

and the Peyer's

patches, while

spread

of

the

infection

to

other

lymphoid

tissues

aswell as

infection of

endothelial and other

epithelial

cells occurs later (lla). It should be remembered that, in

swine, lymph

node tissue is microanatomically inverted in

that

homing

areas of B and Tlymphocytes are interchanged

with T

lymphocytes

located in the lymph node cortex and B

follicles in the medulla (la). Immunocytochemistry

with

anti-IgM

(1) for

staining of

B

lymphocytes

shows an intact

follicle

atthe onset

of

disease (Fig.

4A) and remnants

of

a

destroyed

follicle

at

the terminal

stage

(Fig.

4C). Structural

disorganization

of

lymphoid tissues

has

been

noted in earlier

histopathologic

descriptions of hog

cholera

(15),

butasthis

event wasrecognized

only

late in

disease,

when

coagulopa-thy

and

hemorrhages

had caused

alterations

in a

variety

of

tissues,

acausative role in the

progression

of diseasecould

not

be evaluated and the

histopathology could

not

be

attrib-uted to a

specific cell

tropism

of the virus.

We therefore

consider most

important

the

finding

thatviral RNA localizes

specifically

toBfollicles at a very

early

stage of

hog

cholera

(Fig. 4B),

when the

follicular

structure

is still intact

(Fig.

4A),

clearly

preceding general

leukopenia

and

thymus

atro-phy.

In

situ

hybridization,

with a

mixture

of

radioactively

labeled

ribonucleotides transcribed from

several

HCV

cDNA clones used as a

probe,

demarcates the

central

regions of

a

secondary follicle while

sparing

the dense

mantle zone

(Fig.

4B). This

location of

the HCV genome

suggests

that the

follicle is

the site

of

virus

entry

into the

lymph

node and

unequivocally

demonstrates that it is a

microanatomical

site of virus

replication.

Notably,

attheend

stage

of

disease,

when the

follicular

architecture is

largely

destroyed (Fig.

4C),

viral RNA

is found

condensed,

demar-J. VIROL.

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NOTES 1175

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

stage

of

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

be-cause

of

an

infection

of the supporting follicular dendritic

cell

network.

We plan to address the targetcell question by

trying

to detect HCV gene expression in sorted

developmen-tal stages

of

B lymphocytes from infected swine.

Conclusion. The data have shown that HCV replication in B

follicles is

a specific and early event in hog cholera and

precedes generalized infection. Clearly,depletion of B

lym-phocytes

cannot account for all the pleiotropic

symptoms

of this

disease.

Yet, as

it

is 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 determinant

for 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

acknowledged.

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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Togaviridae.

Inter-virology24:125-139.

VOL.66,1992

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Figure

FIG.1.followedinducedweresamplesinfectiveswineBN, General leukopenia during acute hog cholera
FIG.1.followedinducedweresamplesinfectiveswineBN, General leukopenia during acute hog cholera p.1
FIG. 2.windowsofrepresentantibodyatDickinson, two cells Relative changes in leukocyte subpopulations
FIG. 2.windowsofrepresentantibodyatDickinson, two cells Relative changes in leukocyte subpopulations p.2
FIG. 3.25,000incells,theperipheral the Depletion of B lymphocytes in peripheral blood and lymphoid tissues
FIG. 3.25,000incells,theperipheral the Depletion of B lymphocytes in peripheral blood and lymphoid tissues p.3
FIG. 4.caseC)Calif.).ofhematoxylin-eosinbiotinylatedsections 35S-labeled and Localization of HCV genome in germinal centers in lymph nodes
FIG. 4.caseC)Calif.).ofhematoxylin-eosinbiotinylatedsections 35S-labeled and Localization of HCV genome in germinal centers in lymph nodes p.4

References

Related subjects : Germinal centers hog cholera