JOURNAL OF VIROLOGY, Feb. 1992, p. 1171-1175
Vol. 66, No. 2
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 SUSA,lt MATTHIAS
KONIG,1 ARMIN SAALMULLER,1 MATTHIAS J. REDDEHASE,2 AND HEINZ-JURGEN THIEL'* Federal Research Centre for Virus Diseases of Animals, D-7400 Tubingen,' and Department of Virology,
MILORAD
University of Ulm, D-7900 Ulm,2 Germany Received 6 June 1991/Accepted 4 October 1991
Hog cholera, also known as classical or European swine fever, is caused by hog cholera virus, a member of the genus Pestivirus. It is shown here that the end stage of lethal infection in the natural host is associated with a dramatic depletion preferentially of B lymphocytes in the circulatory system as well as in lymphoid tissues. Already at the onset of disease, viral replication in lymphoid tissues demarcates the germinal centers, and the viral genome remains localized to that site as the disease progresses even after morphologic disintegration of the follicular structure. A block in B-lymphocyte maturation by infection and destruction of germinal centers is discussed as a key event in the pathogenesis of acute, lethal hog cholera.
Early stages in acute hog cholera are associated with fever and diarrhea. The progression of the disease is accompanied by a severe wasting syndrome. The terminal stage is signified by a blue discoloration of the skin and weakness of the hind legs and is sometimes accompanied by neurological symptoms. Disseminated intravascular coagulopathy, extensive tissue hemorrhages, and thymus atrophy are seen at autopsy (for a review, see reference 20). A virus with a positivestranded 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) as well 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 investigators (22) we document that B-lymphocyte deficiency caused by viral destruction of germinal centers in lymphoid tissues is the most significant pathoimmunological consequence 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 *
animals died between days 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 log 50% 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
,110 L 15
*Ly
8 0
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 were made by using the coverslip method of blood film preparation followed by Pappenheim staining. Abbreviations: Mo, monocytes; BN, band form neutrophils; SN, segmented neutrophils; Ly, lymphocytes.
Corresponding author.
t Present address: Department of Virology, University of Ulm, D-7900 Ulm, Germany. 1171
1172
J. VIROL.
NOTES
0dom hhin_2m
Window
Swims
Onset of 1lsem
1
2
3
4
S 0630 S 0631 S 0629 S 0312
4 24 31 14
0
4
39 0 47 49
21
Medbn
27,51
S_im
0D
48 20
2
3
4
28
3
47 LX 31 41
I
S 0630 S 0631 S 0629 S 0312
i
2
46 U
2 4
Medan
36
2.5
1
2
50
3 Q 2 2
End-Stage of mm-
19 25
Window 1
±z
1 1
16
S 0630 S 0631 S 0629 S 0312
78 63
Medin
64.5 2
6i
4
3
20 17 S
17.5
4
31
6
22
i
12 32
5 2
30
4.5
SSC-b FIG. 2. Relative changes in leukocyte subpopulations. Cytofluorometric analyses of peripheral blood leukocytes before infection (top) and at two different stages of disease (center and bottom). Dextran (Ficoll 400)-enriched leukocytes were stained with biotinylated monoclonal antibody 8/1 and allophycocyanin-conjugated streptavidin. The measurements were performed on a dual-laser FACStar Plus (Becton Dickinson, Mountain View, Calif.). In the two-dimensional contour plots of 8/1 antigen expression versus SSC, frames indicate electronic windows set on granulocytes (window 1), monocytes (window 2), T lymphocytes (window 3), and B lymphocytes (window 4). Contour lines represent levels of 5, 10, 20, 40, 60, 80, 100, 120, 140, and 160 cells, with a total of 25,000 cells analyzed. Data in the tables are the percentages of cells located in the respective windows, compiled from data for four individual swine, with the depicted case underlined.
VOL. 66, 1992
NOTES
Peripheral Blood
Spleen
Lymph Node
71
Before Infection
1173
29
fluorescence, .n.e...nluorescenlce intensity
Median
-
Median
-
End- Stage of Disease
21 %
n
-S
Median
-
24 %
n-
3
Median
7%
n
-
S
Median
=
6%
n =
2
Median
90
10
-
=
38 %
n
16 %
n -
89
-
3
2
11
al A4 3......2.......... !i10. fluorescence intensity 0 W0~ .
I1g
.
0
FIG. 3. Depletion of B lymphocytes in peripheral blood and lymphoid tissues. Ficoll-Hypaque-enriched mononuclear leukocytes from peripheral blood, spleen, and mandibular lymph nodes were stained for cytofluorometric analyses with monoclonal antibody 1G6, specific for the porcine Ig kappa chain, and fluorescein isothiocyanate-conjugated F(ab')2 fragments of goat anti-mouse IgG-F(ab')2. For each animal, 25,000 cells were analyzed throughout. The vertical marker delimits the negative control defined by staining with the fluorescent conjugate in the absence of the specific first antibody. The numbers in the upper left and right corners are the percentages of Ig-negative and Ig-positive cells, respectively.
relative changes in peripheral blood leukocyte subpopulations 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, and to the end stage of disease, which was signified by hemorrhages of the skin and a fall in body temperature to below normal. For a random sample of four swine, analyzed individually before infection and during the course of disease, results are compiled in Fig. 2. The porcine leukocyte antigen 8/1 is known to be expressed by all porcine leukocytes with the exception of those of B-lymphocyte lineage (10, 17, 18). In a cytofluorometric analysis, detection of this antigen together with the side scatter (SSC), a physical parameter reflecting cell granularity, allows for a single-step quantitation of the four major leukocyte subpopulations. In the two-dimensional contour plot of 8/1 antigen expression versus SSC (Fig. 2), granulocytes are described as 8/12+SSChi (electronic window 1 in Fig. 2), monocytes are described as 8/13+SSC'° (window 2), resting T lymphocytes are described as 8/1+SSC'° (window 3), and B lymphocytes are described as 8/1-SSC'° (window 4). It is evident from the data that the progression of disease is associated with a significant increase in the proportion of granulocytes and with an almost complete loss of B lymphocytes, whereas the reduction in T lymphocytes is less impressive. In absolute terms, the number of granulocytes
also decreases. However, the augmentation of band form neutrophils over segmented neutrophils (Fig. 1) indicates an increased rate of granulocyte production in response to HCV infection. Alterations within the T-lymphocyte compartment and their significance in disease are the subjects of current research, but it is informative that among the four subpopulations of T lymphocytes known to be present in swine (16), CD4- CD8+ T lymphocytes relatively increase in number, while CD4- CD8- y/8 T lymphocytes (14) are diminished in number and the subpopulations CD4+CD8+ and CD4+CD8are essentially unchanged in peripheral blood (unpublished data). One could raise the objection that the scarcity of B lymphocytes in peripheral blood may result from their recruitment to lymphoid tissue rather than indicate their depletion. We therefore extended the cytofluorometric analysis to lymphoid tissue, represented by the spleen and by mandibular as well as inguinal lymph nodes. Mononuclear leukocytes were enriched, and B lymphocytes were then positively 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 route of infection, intranasal or intramuscular, and was also
1174
,=t ~ .
NOTES
J. VIROL.
T, -
s
.......'....,'
'.
, , ,;, iA
't
AL
, o!:
Vol. 66, No. 2
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 SUSA,lt MATTHIAS
KONIG,1 ARMIN SAALMULLER,1 MATTHIAS J. REDDEHASE,2 AND HEINZ-JURGEN THIEL'* Federal Research Centre for Virus Diseases of Animals, D-7400 Tubingen,' and Department of Virology,
MILORAD
University of Ulm, D-7900 Ulm,2 Germany Received 6 June 1991/Accepted 4 October 1991
Hog cholera, also known as classical or European swine fever, is caused by hog cholera virus, a member of the genus Pestivirus. It is shown here that the end stage of lethal infection in the natural host is associated with a dramatic depletion preferentially of B lymphocytes in the circulatory system as well as in lymphoid tissues. Already at the onset of disease, viral replication in lymphoid tissues demarcates the germinal centers, and the viral genome remains localized to that site as the disease progresses even after morphologic disintegration of the follicular structure. A block in B-lymphocyte maturation by infection and destruction of germinal centers is discussed as a key event in the pathogenesis of acute, lethal hog cholera.
Early stages in acute hog cholera are associated with fever and diarrhea. The progression of the disease is accompanied by a severe wasting syndrome. The terminal stage is signified by a blue discoloration of the skin and weakness of the hind legs and is sometimes accompanied by neurological symptoms. Disseminated intravascular coagulopathy, extensive tissue hemorrhages, and thymus atrophy are seen at autopsy (for a review, see reference 20). A virus with a positivestranded 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) as well 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 investigators (22) we document that B-lymphocyte deficiency caused by viral destruction of germinal centers in lymphoid tissues is the most significant pathoimmunological consequence 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 *
animals died between days 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 log 50% 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
,110 L 15
*Ly
8 0
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 were made by using the coverslip method of blood film preparation followed by Pappenheim staining. Abbreviations: Mo, monocytes; BN, band form neutrophils; SN, segmented neutrophils; Ly, lymphocytes.
Corresponding author.
t Present address: Department of Virology, University of Ulm, D-7900 Ulm, Germany. 1171
1172
J. VIROL.
NOTES
0dom hhin_2m
Window
Swims
Onset of 1lsem
1
2
3
4
S 0630 S 0631 S 0629 S 0312
4 24 31 14
0
4
39 0 47 49
21
Medbn
27,51
S_im
0D
48 20
2
3
4
28
3
47 LX 31 41
I
S 0630 S 0631 S 0629 S 0312
i
2
46 U
2 4
Medan
36
2.5
1
2
50
3 Q 2 2
End-Stage of mm-
19 25
Window 1
±z
1 1
16
S 0630 S 0631 S 0629 S 0312
78 63
Medin
64.5 2
6i
4
3
20 17 S
17.5
4
31
6
22
i
12 32
5 2
30
4.5
SSC-b FIG. 2. Relative changes in leukocyte subpopulations. Cytofluorometric analyses of peripheral blood leukocytes before infection (top) and at two different stages of disease (center and bottom). Dextran (Ficoll 400)-enriched leukocytes were stained with biotinylated monoclonal antibody 8/1 and allophycocyanin-conjugated streptavidin. The measurements were performed on a dual-laser FACStar Plus (Becton Dickinson, Mountain View, Calif.). In the two-dimensional contour plots of 8/1 antigen expression versus SSC, frames indicate electronic windows set on granulocytes (window 1), monocytes (window 2), T lymphocytes (window 3), and B lymphocytes (window 4). Contour lines represent levels of 5, 10, 20, 40, 60, 80, 100, 120, 140, and 160 cells, with a total of 25,000 cells analyzed. Data in the tables are the percentages of cells located in the respective windows, compiled from data for four individual swine, with the depicted case underlined.
VOL. 66, 1992
NOTES
Peripheral Blood
Spleen
Lymph Node
71
Before Infection
1173
29
fluorescence, .n.e...nluorescenlce intensity
Median
-
Median
-
End- Stage of Disease
21 %
n
-S
Median
-
24 %
n-
3
Median
7%
n
-
S
Median
=
6%
n =
2
Median
90
10
-
=
38 %
n
16 %
n -
89
-
3
2
11
al A4 3......2.......... !i10. fluorescence intensity 0 W0~ .
I1g
.
0
FIG. 3. Depletion of B lymphocytes in peripheral blood and lymphoid tissues. Ficoll-Hypaque-enriched mononuclear leukocytes from peripheral blood, spleen, and mandibular lymph nodes were stained for cytofluorometric analyses with monoclonal antibody 1G6, specific for the porcine Ig kappa chain, and fluorescein isothiocyanate-conjugated F(ab')2 fragments of goat anti-mouse IgG-F(ab')2. For each animal, 25,000 cells were analyzed throughout. The vertical marker delimits the negative control defined by staining with the fluorescent conjugate in the absence of the specific first antibody. The numbers in the upper left and right corners are the percentages of Ig-negative and Ig-positive cells, respectively.
relative changes in peripheral blood leukocyte subpopulations 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, and to the end stage of disease, which was signified by hemorrhages of the skin and a fall in body temperature to below normal. For a random sample of four swine, analyzed individually before infection and during the course of disease, results are compiled in Fig. 2. The porcine leukocyte antigen 8/1 is known to be expressed by all porcine leukocytes with the exception of those of B-lymphocyte lineage (10, 17, 18). In a cytofluorometric analysis, detection of this antigen together with the side scatter (SSC), a physical parameter reflecting cell granularity, allows for a single-step quantitation of the four major leukocyte subpopulations. In the two-dimensional contour plot of 8/1 antigen expression versus SSC (Fig. 2), granulocytes are described as 8/12+SSChi (electronic window 1 in Fig. 2), monocytes are described as 8/13+SSC'° (window 2), resting T lymphocytes are described as 8/1+SSC'° (window 3), and B lymphocytes are described as 8/1-SSC'° (window 4). It is evident from the data that the progression of disease is associated with a significant increase in the proportion of granulocytes and with an almost complete loss of B lymphocytes, whereas the reduction in T lymphocytes is less impressive. In absolute terms, the number of granulocytes
also decreases. However, the augmentation of band form neutrophils over segmented neutrophils (Fig. 1) indicates an increased rate of granulocyte production in response to HCV infection. Alterations within the T-lymphocyte compartment and their significance in disease are the subjects of current research, but it is informative that among the four subpopulations of T lymphocytes known to be present in swine (16), CD4- CD8+ T lymphocytes relatively increase in number, while CD4- CD8- y/8 T lymphocytes (14) are diminished in number and the subpopulations CD4+CD8+ and CD4+CD8are essentially unchanged in peripheral blood (unpublished data). One could raise the objection that the scarcity of B lymphocytes in peripheral blood may result from their recruitment to lymphoid tissue rather than indicate their depletion. We therefore extended the cytofluorometric analysis to lymphoid tissue, represented by the spleen and by mandibular as well as inguinal lymph nodes. Mononuclear leukocytes were enriched, and B lymphocytes were then positively 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 route of infection, intranasal or intramuscular, and was also
1174
,=t ~ .
NOTES
J. VIROL.
T, -
s
.......'....,'
'.
, , ,;, iA
't
AL
, o!:
