Thymocyte and Peripheral Blood T Lymphocyte Subpopulation Changes in Piglets Following
in Utero Infection with Porcine Reproductive and Respiratory Syndrome Virus
Wen-hai Feng,*,1M. B. Tompkins,† Jin-Sheng Xu,* T. T. Brown,† S. M. Laster,‡ He-xiao Zhang,§ and M. B. McCaw*,2
*Department of Farm Animal Health and Resource Management, †Department of Microbiology, Pathology, and Parasitology, College
of Veterinary Medicine, ‡Department of Microbiology, College of Agriculture and Life Sciences, North Carolina State University, Raleigh,
North Carolina 27606; and §Quarantine Section, Beijing Entry-Exit Inspection and Quarantine Bureau, #12 Jian Guo Men Wai Street,
Chaoyang District, Beijing 100022, China
Received December 7, 2000; returned to author for revision February 20, 2001; accepted May 8, 2002
Piglets infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV) are born severely immuno-
compromised. In this article we more closely examine the effects of in utero PRRSV infection on circulating and thymic T cell
populations. Numbers of CD4?, CD8?, and dual-positive lymphocytes were quantitated in circulation and in the thymus during
the 2 weeks following birth. At birth we found that the number of circulating lymphocytes was suppressed by 60%.
Lymphocyte numbers were also suppressed by 42% at day 7, but by day 14 the number of lymphocytes had rebounded and
was actually 47% greater than controls. At birth and day 7, a drop in the number of CD4?cells could partially explain the
suppression we observed, while the rebound in total lymphocyte numbers seen at day 14 was due to a nearly fourfold
increase in the number of circulating CD8?cells. As a result, the normal CD4?:CD8?ratio of between 1.4 and 2.2 for neonatal
pigs was reduced to 0.1–0.5. The thymuses of infected piglets were found to be 50% smaller than those of control pigs and
were characterized by cortical involution and severe cortical depletion of thymocytes. Analysis of the population of
thymocytes revealed that double-positive thymocytes were suppressed to a greater degree than either single positive
subpopulation. In addition, we show that the number of thymocytes undergoing apoptosis was increased twofold in piglets
infected with PRRSV. Taken together, these results help explain the dramatic immunosuppression observed in neonatal
animals infected in utero with PRRSV.
© 2002 Elsevier Science (USA)
Key Words: PRRSV; thymus; thymocyte; T lymphocyte.
Porcine reproductive and respiratory syndrome (PRRS)
is a viral disease of swine (Terpstra et al., 1991; Wens-
voort et al., 1991) that was first recognized in the United
States in 1987 and in Europe in 1990 (Keffaber, 1989;
Terpstra et al., 1991; Wensvoort et al., 1991). The etiologic
agent first isolated in 1991 is a single-stranded RNA virus
(Benfield et al., 1992; Wensvoort et al., 1991). Porcine
reproductive and respiratory syndrome virus (PRRSV) is
classified as a member of the genus Arterivirus, family
Arteriviridae, which includes lactate dehydrogenase ele-
vating virus (LDV), equine arteritis virus (EAV), and simian
hemorrhagic fever virus (SHFV) (Conzelmann et al., 1993;
Meulenberg et al., 1993). The genome of PRRSV is 15 kb
in length, which contains eight open reading frames
(ORFs). Of the structural genes (ORFs 2 through 7), ORF
7 is known to code for the nucleocapsid protein, and
ORFs 2, 3, 4, 5, and 6 code for envelope proteins (Meu-
lenberg et al., 1996). PRRSV can infect pigs in all stages
of development. It can cause severe respiratory distress,
fever, lethargy, and high mortality in suckling, weaned,
and grow-finish pigs (Keffaber, 1989; Collins et al., 1992;
Wensvoort et al., 1991), and late-term abortions, and
mummified, stillborn, and weak-borne pigs in pregnant
sows (Christianson et al., 1992).
Field observations suggest that PRRSV can also cause
immunosuppression. PRRSV-infected animals often de-
velop secondary infections with bacteria (Done and Pa-
ton, 1995). However, it has been difficult to experimen-
tally re-create the immunosuppressive aspects of
PRRSV. Galina et al. (1994) first reported that PRRSV
infection could predispose pigs to more severe disease
and mortality following infection with Streptococcus suis
(MN 87555) compared with those infected with either
PRRSV or S. suis alone. Wills et al. (2000) demonstrated
an increase in severity of disease when PRRSV chal-
lenge followed Salmonella choleraesuis infection and
dexamethasone treatment. Also, Reeth et al. (1996) re-
ported a positive association of PRRSV with porcine
respiratory coronavirus or swine influenza virus. How-
ever, Cooper et al. (1995) reported that infection with
1Current address: Linberger Comprehensive Cancer Center, CB#
7295, University of North Carolina at Chapel Hill, UNC-Chapel Hill,
Chapel Hill, North Carolina 27599.
2To whom correspondence and reprint requests should be ad-
dressed at Department of Farm Animal Health and Resource Manage-
ment, College of Veterinary Medicine, 4700 Hillsborough Street, Ra-
leigh, North Carolina 27606. Fax: (919) 513-6464. E-mail: Monte_
Virology 302, 363–372 (2002)
© 2002 Elsevier Science (USA)
All rights reserved.
NEB-1 strain of PRRSV did not exacerbate disease when
infection was followed 2 or 7 days later with Haemophi-
lus parasuis, S. suis, S. choleraesuis, or Pasteurella mul-
tocida infection. Others (Carvalho et al., 1997; Pol et al.,
1997; Solano et al., 1997) also reported failed attempts to
re-create secondary bacteria infection following PRRSV
Recently, we have reported on a new model system
that accurately reproduces the immunosuppressive ef-
fects of PRRSV (Feng et al., 2001). Sows were infected at
day 98 of gestation and the resulting neonates were
found to be highly susceptible to a normally nonlethal
dose of S. suis. Lesions were also observed in a variety
of organs. Most striking was the thymus, which dis-
played a nearly complete lack of cortical thymocytes. In
this article we expand on our investigations of neonates
that have received PRRSV in utero and more closely
examine both circulating and intrathymic T cell popula-
tions. Our results reveal dramatic suppression of circu-
lating CD4?T cells, while CD8?levels actually increase
above levels seen in control animals, as well as signifi-
cant changes in thymocyte subpopulations.
The animals examined in this study were infected with
PRRSV in utero at day 98 of gestation. We found this to be
a highly effective method for transmitting the virus. In-
fectious PRRSV was detected in the sera of all piglets
(9/9) at 0, 7, and 14 days of age (Table 1). Infectious
PRRSV was also detected in the thymuses of all piglets
at each time point, except for one piglet on day 0 and two
piglets on day 14. All control piglets remained PRRSV
Lymphocyte numbers in peripheral blood
As shown in Fig. 1, we found a significant decrease in
the number of peripheral blood lymphocytes in PRRSV-
infected piglets at 0 and 7 days of age. Lymphocytes
were decreased by 60% at birth and by 42% at day 7. The
decrease in peripheral lymphocytes seen at these time
points could be attributed partly to a drop in the number
of circulating CD4?T lymphocytes (Fig. 2A). CD4?cells
were decreased by approximately 450 cells/ml at birth
and 1400 cells/ml at day 7, accounting for 41 and 46%,
respectively, of the drop in total lymphocyte levels. On a
population basis, in control animals we found that CD4?
T cells make up 41 and 29% of total peripheral lympho-
cytes at days 0 and 7, respectively (Fig. 3A). This per-
centage dropped to 15 and 19%, respectively, in PRRSV-
infected animals. In contrast, at day 0 we did not find a
significant change in the number of CD8?T cells (Fig.
2B), even though the percentage of CD8?T cells was
significantly increased (Fig. 3B). However, at day 7, the
number of CD8?T cells was significantly increased (Fig.
2B), in coordinate with the significantly increased per-
centage of CD8?T cells (Fig. 3B). Pigs usually have a
relatively high percentage of CD4?CD8?dual expression
peripheral T cells (Pescovitz et al., 1994). In PRRSV-
Detection of PRRSV in the Sera and Thymuses of Infected Piglets at 0, 7, and 14 Days of Age by Virus Isolationa
Day 0 Day 7Day 14 Day 0Day 7Day 14
Note. Data are expressed as number of positive piglets/total piglets examined.
aPRRSV isolation was performed by inoculation of serum or thymic tissue homogenate onto primary porcine alveolar macrophage cultures.
Confirmation of PRRRV isolation was performed by FA testing using SDOW-17 Mab against PRRSV nucleoprotein.
bInfected treatment piglets were born to PRRSV-infected sows and control piglets were collected from uninfected sows.
FIG. 1. Numbers of lymphocytes in peripheral blood (PB) from PRRSV-
infected and noninfected piglets at 0, 7, and 14 days of age. Bar
represents mean ? standard deviation. Eight pigs were examined for
each treatment group, except nine pigs in the infected 0-day-old treat-
ment group. Infected piglets were born to PRRSV-infected sows and
control piglets from uninfected sows. * Significant difference (P ?
0.05) from age-matched control piglets using Student’s t-test.
364 FENG ET AL.
infected piglets, the percentage of CD4?CD8?T cells
was significantly increased over that of control piglets at
day 0, 7, and 14 (Fig. 3C).
The results were quite different on day 14. While the
number of CD4?T cells remained depressed (Fig. 2A),
the total number of lymphocytes was actually increased
by 80% (Fig. 1). As shown in Fig. 2B, the increase in total
lymphocytes was due to a large increase in the number
of circulating CD8?T cells. The cumulative effect of
these changes resulted in a significant decrease in pe-
ripheral CD4?/CD8?T cell ratios in PRRSV-infected pig-
lets (Fig. 3D). In uninfected control animals we observed
the ratio to decrease from 2.2:1 at birth to 1.5:1 and 1.4:1
at days 7 and 14, respectively. Animals infected with
PRRSV had CD4?/CD8?ratios of 0.5, 0.4, and 0.1 at days
0, 7, and 14, respectively.
We have reported previously that neonates infected
with PRRSV in utero display thymic abnormalities (Feng
et al., 2001). Similar results were found for the animals in
this investigation. Histologically, PRRSV-infected piglets
consistently exhibited thymic lesions characterized by
depletion of thymocytes, causing a loss of distinction
between cortical and medullary zones in the thymus (Fig.
4). Thymic weights were determined to confirm these
observations. As shown in Table 2, thymic weights were
reduced by 50% at birth and by more than two-thirds at
days 7 and 14, respectively. We estimate that the number
of thymocytes was suppressed by approximately 75%
based upon the more than 50% reduction recorded in
thymic weight and the observed marked reduction in
density of thymocytes (Fig. 4).
Next, we investigated whether the loss of thymocytes
was selective for one subpopulation over another. Anal-
ysis of individual thymocyte subsets defined by CD4 and
CD8 expression showed that the proportion of CD4?and
CD8?subpopulations in the thymus was comparable
between control and virus-infected animals at 0, 7, and
14 days of age (Fig. 5A and 5B), except for a significant
increase in the percentage of CD4?CD8?thymocytes
observed at birth (Fig. 5B). The number of CD4?and
CD8?cells in the thymus of PRRSV-infected animals was
estimated based on thymus weight and cellularity of
thymocytes and both were reduced by 70–75%. A signif-
icant reduction in the proportion of CD4?CD8?was ob-
served (Fig. 5C). These cells, which normally comprise
60–70% of thymocytes, were reduced disproportionately
in PRRSV-infected animals so that they accounted for
only 40–50% of the total number of thymocytes. The
ratios of CD4?/CD8?thymocytes were similar in control
and PRRSV-infected piglets, except a significant de-
crease in virus-infected piglets at birth.
It has been shown that PRRSV infection may initiate
programmed cell death in vivo and in vitro (Suarez et al.,
1996; Sur et al., 1997; Sirinarumitr et al., 1998). Therefore,
we investigated whether apoptosis is associated with
thymocyte depletion in in utero PRRSV-infected piglets.
The frequency of apoptotic cells in normal and in utero
PRRSV-infected piglet thymus was analyzed by flow cy-
tometry using TUNEL method. The results revealed a
doubling of apoptotic thymocytes in PRRSV-infected pig-
lets over that of controls (Table 3).
This study demonstrates that in utero infection with
PRRSV of piglets resulted in infection of cells within the
thymus which is accompanied by thymic atrophy, pro-
found thymocyte depletion, and increased rate of apo-
ptosis. Virus-mediated interference with thymopoiesis
may contribute to the pathogenesis of PRRSV infection.
Examples of virus-induced thymic involution are human
FIG. 2. Numbers of CD4?and CD8?cells in peripheral blood (PB)
from PRRSV-infected and noninfected piglets at 0, 7, and 14 days of age.
Lymphocyte subsets were analyzed by two-color flow cytometry. (A)
Number of CD4?CD8?cells in PB. The number of CD4?CD8?cells in
PRRSV-infected piglets was significantly decreased compared to con-
trol piglets at 0, 7, and 14 days of age. (B) Number of CD4?CD8?cells
in PB. The number of CD4?CD8?cells in PRRSV-infected piglets were
significantly increased compared to control piglets at 7 and 14 days of
age. Data points represent mean ? standard deviation. Eight pigs were
examined for each treatment group, except nine pigs in the infected
treatment group at 0 days of age. * Significant difference between
PRRSV-infected and noninfected pigs (P ? 0.05).
365T LYMPHOCYTE SUBPOPULATION CHANGES IN PIGLETS
immunodeficiency virus in humans (Bonyhadi et al.,
1993), feline immunodeficiency virus (FIV) in cats (Beebe
et al., 1994), lactate dehydrogenase-elevating virus in
mice (Plagemann et al., 1995), mouse hepatitis virus in
mice (Godfraind et al., 1995), swine fever virus in pigs
(Sato et al., 2000), and murine cytomegalovirus in mice
(Price et al., 1993). Our interest in the thymic effects of in
utero PRRSV infection was a result of prior studies which
documented the ability of PRRSV to infect thymus (Ros-
sow et al., 1995; Halbur et al., 1995, 1996) and the in-
creased susceptibility to secondary infections of PRRSV-
infected pigs (Done and Patton, 1995; Feng et al., 2001).
Marked pathologic changes of the thymus, character-
ized by thymic atrophy, were observed in piglets infected
in utero with PRRSV. Thymic atrophy associated with
obvious cortical involution was evident during the first 2
weeks of life. The profound loss of cortical thymocytes in
piglets infected with PRRSV in utero was similar to find-
ings described for other neonatal viral infections (John-
son et al., 1998; Rosenzweig et al., 1993; Parsonson et al.,
1977). Johnson et al. reported that kittens directly in-
fected with feline immunodeficiency virus at 6 weeks of
gestation displayed severe thymus atrophy, which was
characterized by expanded interlobular spaces and re-
duced thymocyte density 4 weeks postinoculation. Par-
sonson et al. reported that infection of pregnant sheep
with Akabane virus induced depletion of thymocytes in
newborn lambs. These structural changes noted in the
thymus of newborn piglets suggest that thymic function
was compromised. In contrast, pathologic changes of
the thymus were not observed in pigs infected with
PRRSV after birth (Rossow et al., 1994). In their report, 1-,
4-, and 10-week-old pigs were infected with ATCC VR-
2332 PRRSV. No thymic lesions were reported at 7 or 28
days postinfection. PRRSV was isolated from thymic tis-
sues of all the 1-week-old pigs and one-third of the 4-
and 10-week-old pigs after inoculation, suggesting age
plays a role in the pathogenesis of PRRSV infection.
Possibly, in the early stages of development of the thy-
mus, thymic tissues and cells may be more susceptible
to PRRSV infection.
The potential impact of PRRSV infection on thymocyte
FIG. 3. Percentage of lymphocyte subsets in peripheral blood of PRRSV-infected and noninfected piglets at 0, 7, and 14 days of age. CD4?CD8?
and CD4?CD8?cells were analyzed by two-color cytometry. (A) Percentage of CD4?CD8?T lymphocytes in PRRSV-infected and noninfected control
pigs. (B) Percentage of CD4?CD8?T lymphocytes in PRRSV-infected and noninfected control pigs. (C) Percentage of CD4?CD8?T lymphocytes in
PRRSV-infected and noninfected control pigs. (D) CD4?:CD8?ratio of T lymphocytes in PRRSV-infected and noninfected control pigs. Bar represents
mean values ? standard deviation. Eight pigs were examined for each treatment group, except nine pigs in the infected treatment group on day 0.
* Significant difference between PRRSV-infected and noninfected pigs (P ? 0.05).
366FENG ET AL.
maturation events was assessed by examination of CD4
and CD8 expression on thymocyte subsets. We observed
an apparent decrease in the total number of thymocytes
in in utero PRRSV-infected piglets compared to control
piglets. The percentage of CD4?CD8?and CD4?CD8?
thymocytes did not change significantly, except for a
significant increase in CD4?CD8?thymocyte subset at 0
day. However, the percentage of the CD4?CD8?double-
positive thymocytes was significantly decreased in all
age groups of piglets infected in utero with PRRSV.
These findings suggest that PRRSV causes a significant
loss of CD4?CD8?thymocytes, but may not affect further
differentiation of these cells into either CD4?CD8?or
CD4?CD8?lymphocytes. This was not observed in pigs
infected with PRRSV after birth (Shimizu et al., 1996). In
that study, 5-day-old colostrum-deprived pigs were in-
fected with PRRSV and the thymocytes were analyzed
after 1, 2, 4, and 6 weeks. There were no differences in
thymocyte subpopulations observed in that study be-
tween infected and noninfected pigs. Our results were
similar to findings described in HIV infection (Su et al.,
1995), FIV infection (Johnson et al., 1998), and mouse
hepatitis virus (Godfraind et al., 1995), for which thymic
depletion of CD4?CD8?immature thymocytes was de-
We examined the frequency of apoptotic cells in nor-
mal and in utero PRRSV-infected piglet thymus, since
programmed cell death is an integral feature of normal
thymocyte development. In our study the loss of thymo-
cytes appears to be mediated at least in part by pro-
grammed cell death. Suarez et al. (1996) had previously
demonstrated that the glycoprotein p25, encoded by
PRRSV ORF 5, induces apoptosis when expressed in
COS-1 cells. Apoptosis also occurred in PRRSV-infected
cells (MA-104 and swine alveolar macrophages). Later
Sur et al. (1997) reported that PRRSV can also infect and
induce testicular germ cell death by apoptosis. Sur et al.
(1998) and Sirinarumitr et al. (1998) reported that PRRSV
infection resulted in widespread apoptosis in lungs and
lymphoid tissues. Now our observation of the increased
apoptotic cells in thymus in in utero infected piglets
emphasizes the biological relevance of this property of
Several mechanisms might be proposed to account for
the PRRSV-mediated thymocyte depletion in in utero
PRRSV-infected piglet model. First, viral infection may kill
thymocytes or their progenitors, either directly of indi-
rectly. Several viruses have been shown to induce apo-
ptosis either directly (Jolicoeur and Lamontagne, 1994;
Lamontagne and Jolicoeur, 1991; Lu et al., 1996) or indi-
rectly (Finkel et al., 1995; Goldfraind et al., 1995). Since
PRRSV antigen was not detected in thymocytes, viral
infection of thymic lymphocytes might not be the cause
of PRRSV-induced thymic involution. However, apoptosis
in the course of viral infection can result from the secre-
tion of soluble molecules. Several cytokines, such as
TNF? and IL-1?, have been shown to promote apoptosis
of T cell lines. In the course of PRRSV infection, alveolar
macrophages have been shown to express high levels of
IL-1? (Zhou et al., 1992), which might also be the case in
PRRSV-infected thymic macrophages and dendritic cells
and may play a role in thymocyte depletion. Additionally,
Thymic Weights (g) from PRRSV-Infected and Noninfected Control
Piglets at 0, 7, and 14 Days of Age
Day 0 Day 7Day 14
1.41 ? 0.69*
2.84 ? 0.60
2.98 ? 0.79*
10.74 ? 3.53
8.73 ? 3.19*
29.43 ? 8.41
Note. Data are expressed as mean thymic weights ? standard
aInfected treatment piglets were born to PRRSV-infected sows and
control piglets were collected from uninfected sows. Eight pigs were
examined for each treatment group, except nine in the infected 0-day-
old treatment group.
* Significantly different (P ? 0.05) from age-matched control piglets
by Student’s t-test.
FIG. 4. Thymic atrophy in a 7-day-old piglet infected in utero with
PRRSV (hematoxylin and eosin stain). (A) A thymus of age-matched
control piglet, showing the distinctive corticomedullary junction. (B)
Marked depletion of cortical thymocytes resulting in loss of cortico-
medullary definition in a PRRSV-infected piglet. Magnification, ?350.
367T LYMPHOCYTE SUBPOPULATION CHANGES IN PIGLETS
it has been shown that the production of superoxide
anion was significantly decreased by PRRSV-infected
macrophages. This could be related to apoptosis induc-
tion of the virus, since Sindbis virus is able to activate
apoptosis by reducing the intracellular superoxide levels
(Lin et al., 1999). The p25 protein of PRRSV was demon-
strated to induce apoptosis (Suarez et al., 1996) and the
release of this protein from the infected thymic macro-
phages and dendritic cells could induce thymocyte apo-
ptosis. PRRSV-induced apoptosis in bystander cells was
reported (Sirinarumitr et al., 1998; Sur et al., 1997, 1998).
This indicates that there are indirect mechanisms in-
volved in PRRSV-induced apoptosis. Second, PRRSV in-
fection of thymic macrophages, dendritic cells, and pos-
sibly epithelial cells may compromise their functions in
regulating thymopoiesis. Thymic macrophages, dendritic
cells, and epithelial cells play a central role in T cell
maturation by driving development from CD4?CD8?dou-
ble-negative precursors through CD4?CD8?double-pos-
itive to mature CD4?CD8?or CD4?CD8?thymocytes
(Rothemberg, 1992). It was reported that PRRSV repli-
cates in thymic macrophages and dendritic cells (Halbur
FIG. 5. Percentage of thymocyte subsets in PRRSV-infected and noninfected piglets at 0, 7, and 14 days of age. CD4?CD8?and CD4?CD8?cells
were analyzed by two-color cytometry. (A) Percentage of CD4?CD8?thymocytes in PRRSV-infected and noninfected control pigs. (B) Percentage of
CD4?CD8?thymocytes in PRRSV-infected and noninfected control pigs. (C) Percentage of CD4?CD8?thymocytes in PRRSV-infected and noninfected
control pigs. (D) CD4?:CD8?ratio of thymocytes in PRRSV-infected and noninfected control pigs. Bar represents mean values ? 1.0 standard
deviation. Eight pigs were examined for each treatment group, except nine pigs in the infected treatment group on day 0. * Significant difference
between PRRSV-infected and noninfected pigs using Student’s t-test (P ? 0.05).
Thymocytes Undergoing Apoptosis Detected by TUNEL in PRRSV-
Infected and Noninfected Control Piglets at 0, 7, and 14 Days of Age
Day 0 Day 7 Day 14
5.92 ? 2.53*
2.23 ? 1.14
8.24 ? 3.03*
3.79 ? 1.76
14.32 ? 4.64*
6.72 ? 3.10
Note. Data are expressed as mean percentage (%) ? standard
deviation of thymocyte population.
aInfected treatment piglets were born to PRRSV-infected sows and
control piglets were collected from uninfected sows. Eight pigs were
examined for each treatment group, except nine pigs in the infected 0-
day-old treatment group.
* Significant difference (P ? 0.05) from age-matched control piglets
368 FENG ET AL.
et al., 1996). PRRSV was also detected in nasal epithelial
cells, nasal serous gland epithelial cells, and cells lining
airways (Pol et al., 1991; Rossow et al., 1996; Thanawong-
nuwech et al., 1997). These observations suggest that
thymic epithelial cells might also be the target of PRRSV
infection. Infections of these cells, resulting in lysis or in
impairment of their activity, might lead to thymocyte dis-
appearance, which is suspected to be responsible for
the increased susceptibility to apoptosis found in dou-
ble-positive thymocytes after inoculation with mouse
hepatitis virus (Lamontagne and Jolicoeur, 1991), murine
cytomegalovirus (Koga et al., 1994), and HIV (Maroder et
al., 1996). Finally, prethymic progenitor cells in bone
marrow may be depleted by PRRSV. PRRSV antigen was
not detected in bone marrow. However, abnormalities,
such as bone marrow hypoplasia characterized by an
absence of normal myeloid and erythroid precursors,
have been described in in utero PRRSV-infected piglets
(Feng et al., 2001). It is possible that in PRRSV-infected
piglets hematopoietic progenitors (including CD34?
cells) are depleted and the animals fail to produce a
sufficient number of progenitor cells to target them to the
In utero PRRSV infection resulted in morphologic al-
terations, changes in thymocyte subpopulations, and
PRRSV infection of cells in the thymus, suggesting thymic
function and thymocyte maturation might be severely
impaired. These observations are significant, since thy-
mic impairment could affect the replenishment of mature
T cells into the peripheral blood, which could partially
explain the lymphocytopenia observed at 0 and 7 days of
age in piglets.
Changes in peripheral blood lymphocyte subpopula-
tions were also observed in addition to alterations in
total lymphocyte numbers following in utero infection
with PRRSV. Our results agree with the results reported
previously (Albina et al., 1998; Christianson et al., 1993;
Shimizu et al., 1996). Shimizu et al. reported 5-day-old
SPF pigs infected with PRRSV revealed decreases in
CD4?cells and the ratios of CD4?/CD8?cells. The de-
cline of CD4?cells continued for at least 14 days and
CD8?cells slightly decreased in number on postinocu-
lation day 3 and then increased markedly. Christianson
et al. (1993) also observed the decline in the ratios of
CD4?/CD8?cells in sows infected with PRRSV. Albina et
al. reported that there was a decrease in the ratios of
CD4?/CD8?cells and an increase in the number of CD8?
cells in 8-week-old pigs infected with PRRSV. However,
the decline of CD4?was not observed in their report. It
was reported that the cell-mediated immune responses
against PRRSV involved mainly CD4?cells (Bautista and
Molitor, 1997). The depression of CD4?cells in our
model might play a role in the immunosuppression of
infected piglets. The significance of the increase in CD8?
T cell numbers in infected pigs is not well understood.
Some investigators suggested that CD8?cells could play
a role in the control of virus replication (Albina et al.,
1998; Samsom et al., 2000). However, this issue remains
to be elucidated. The significance and possible mecha-
nisms of the decrease of the CD4?/CD8?cell ratio in
PRRSV-infected pigs need to be investigated. Our results
also showed that the percentage of CD4?CD8?dual-
expressing peripheral T cells significantly increased in
piglets infected in utero with PRRSV. The porcine T cell
population is unique in that there is a large percentage of
CD4?CD8?dual-expressing T cells (Pescovitz et al.,
1994). However, we still do not know the biological im-
portance of these dual-positive cells in the peripheral
In summary, our study shows that in utero infection
with PRRSV can induce thymic involution, apoptosis of
thymocytes, and disorder of peripheral blood T cell sub-
populations in piglets for at least the first 2 weeks of life,
suggesting the thymic function and immune response of
piglets may be modulated. The in utero PRRSV-infected
piglet model may provide valuable insights into the
mechanisms associated with PRRSV pathogenesis. To
further understand the pathogenesis of in utero infection
with PRRSV, it will be important to investigate the mech-
anisms associated with PRRSV-induced thymocyte de-
pletion and evaluate the effect of in utero PRRSV infec-
tion on the cytokine profiles produced by thymocytes and
peripheral blood T cells.
MATERIALS AND METHODS
Animals and experimental design
Eight PRRSV-free pregnant sows were purchased from
an isolated herd, which had never been vaccinated, was
free of clinical signs of PRRSV infection, and had re-
mained seronegative to PRRSV for the past 3 years. Five
sows were inoculated with strain SD 23983 of PRRSV (a
gift from Dr. Benfield, South Dakota State University) at
98 days gestation by intranasal injection with 2 ml of 103.2
TCID50/ml virus solution as previously described (Kim et
al., 1993). Three other sows served as noninfected con-
trols. All sows were allowed to farrow naturally. Piglets
were deprived of colostrum and removed from their
dams at birth. Piglets were placed into individual cages
in either PRRSV-infected or control rooms and fed milk
replacer (Gomez et al., 1995). Groups of approximately
eight piglets each from PRRSV-infected or control unin-
fected sows were euthanized at 0, 7, and 14 days after
Blood and tissues samples
Prior to euthanasia, peripheral blood was collected
from each piglet into tubes containing EDTA as an anti-
coagulant for complete blood count (CBC) and flow cy-
tometric analysis of lymphocyte subsets. At necropsy, the
thymuses dissected from each pig were weighed and
369T LYMPHOCYTE SUBPOPULATION CHANGES IN PIGLETS
placed in ice-cold PBS supplemented with 2% fetal bo-
vine serum (FBS). Pieces of thymic tissue were fixed in
10% buffered neutral formalin for routine histopathology.
Thymocyte cell suspensions were prepared by pressing
the tissue through a sieve in ice-cold PBS supplemented
with 2% FCS (Aspinall, 1997).
Monoclonal antibodies (MAbs) and reagents
MAbs specific for porcine leukocyte antigens were
used to label cells for flow cytometric analysis. Antipor-
cine CD4 MAb (MAb 74-12-4, VMRD, Inc., Pullman, WA)
was conjugated with fluorescein isothiocyanate (FITC).
Porcine CD8 MAb (MAb 76-2-11, VMRD, Inc.) was biotin-
ylated (a generous gift from Dr. F. A. Zuckermann, Uni-
versity of Illinois at Urbana-Champaign). Biotinylation of
the CD8 MAb permitted the use of R-phycoerythrin (RPE)-
conjugated streptavidin (DAKO, Carpinteria, CA). Fluo-
rescent isothiocyanate-conjugated MAb against PRRSV
nucleoprotein, SDOW 17, was used for virus detection
(Nelson et al., 1993).
Virus isolation from serum or thymic tissues was per-
formed by coculture of serum samples or thymic tissue
homogenate with uninfected porcine alveolar macro-
phages (PAMS) as described previously (Rossow et al.,
1994, 1995). Cultures not displaying cytopathic effect
after two passages were considered negative. PRRSV-
induced cytopathic effect was confirmed using SDOW 17
MAb fluorescein conjugate against PRRSV nucleoprotein
for immunofluorescent staining of PAMS collected from
sample cultures (Wills et al., 1997).
For the analysis of thymocyte subpopulations defined
by their expression of CD4 and CD8, cells were stained
with anti-CD4-FITC, biotin-conjugated anti-CD8, or con-
trol antibody conjugated to FITC or biotin. After washing,
cell preparations were subsequently incubated with
RPE-conjugated streptavidin. Stained cells were ana-
lyzed with a FACScan analytical cytometer (Becton–Dick-
inson, San Jose, CA).
The distribution of T lymphocyte subpopulations was
determined by flow cytometry. For the analysis of blood
lymphocytes, 100 ?l of blood was stained with anti-CD4-
FITC and biotin-conjugated anti-CD8 or the control anti-
body conjugated to FITC or biotin. After washing, cells
were subsequently incubated with RPE-streptavidin.
Erythrocytes were lysed after staining by adding 2 ml
FACS lysing solution (Becton–Dickinson) to each sample.
The cells were analyzed with FACScan analytical cytom-
eter (Becton–Dickinson). The absolute number of CD4?
and CD8?cells was obtained based on the percentages
of each subset and the number of lymphocytes/per mil-
liliter in the blood.
Thymocyte apoptosis detected by TUNEL staining
Detection of thymocyte apoptosis was performed by
the TUNEL technique as previously reported (Sgonc et
al., 1994) following the In Situ Cell Death Detection Kit’s
procedure (Boehringer Mannheim, Indianapolis, IN).
Briefly, thymocytes were fixed with 4% freshly prepared
paraformaldehyde in PBS for 30 min. After washing, cells
were permeabilized by using permeabilization solution
(0.1% Triton X-100 in 0.1% sodium citrate). Cells were
subsequently stained by TUNEL reaction mixture con-
taining the terminal deoxynucleotidyl transferase and
fluorescein-dUTP. Stained cells were analyzed with a
FACScan analytical cytometer.
Statistical analysis was carried out using the Student’s
We thank Dr. Zuckermann for the generous gift of biotinylated por-
cine CD8 Mab and Dr. Benfield for the SD28983 PRRSV strain. We also
thank Dr. Jack Britt for support through this project. This research was
funded by North Carolina Pork Producers Association, College of Vet-
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