Arch Virol DOI 10.1007/s00705-015-2401-7
ORIGINAL ARTICLE
A study of lymphoid organs and serum proinflammatory cytokines in pigs infected with African swine fever virus genotype II Hovakim Zakaryan1 • Victorya Cholakyans1 • Lusine Simonyan1 • Alla Misakyan1 Elena Karalova1 • Andranik Chavushyan2 • Zaven Karalyan1
•
Received: 5 December 2014 / Accepted: 15 March 2015 Ó Springer-Verlag Wien 2015
Abstract African swine fever virus (ASFV), the causative agent of one of the most important viral diseases of domestic pigs for which no vaccine is available, causes immune system disorders in infected animals. In this study, the serum levels of proinflammatory cytokines, as well as the histological and cellular constitution of lymphoid organs of pigs infected with ASFV genotype II were investigated. The results showed a high degree of lymphocyte depletion in the lymphoid organs, particularly in the spleen and lymph nodes, where ASFV infection led to a twofold decrease in the number of lymphocytes on the final day of infection. Additionally, ASFV-infected pigs had atypical forms of lymphocytes found in all lymphoid organs. In contrast to lymphocytes, the number of immature immune cells, particularly myelocytes, increased dramatically and reached a maximum on day 7 postinfection. The serum levels of TNF-a, IL-1b, IL-6, and IL-8 were evaluated. Proinflammatory cytokines showed increased levels after ASFV infection, with peak values at 7 days postinfection, and this highlights their role in the pathogenesis of ASFV. In conclusion, this study showed that ASFV genotype II, like other highly virulent strains, causes severe pathological changes in the immune system of pigs.
& Zaven Karalyan [email protected] 1
Laboratory of Cell Biology and Virology, Institute of Molecular Biology of NAS RA, 0014 Yerevan, Armenia
2
Laboratory of Human Genomics and Immunomics, Institute of Molecular Biology of NAS RA, 0014 Yerevan, Armenia
Introduction African swine fever (ASF) is a highly contagious disease affecting domestic pigs and wild suids. In acute forms of this disease, symptoms in domestic pigs include fever, vascular changes (particularly bleeding), cyanosis of the skin, abdominal pain, and diarrhoea, which may become bloody prior to death [1, 17, 19]. These clinical signs are accompanied by severe thrombocytopenia and lymphopenia. Pigs usually die in less than 10 days. Due to its high mortality rate, which approaches 100 %, and the lack of vaccines, ASF is one of the most important viral diseases of domestic pigs. African swine fever virus (ASFV), the causative agent of ASF, is a large, enveloped double-stranded DNA virus. It is the only member of the genus Asfivirus within the family Asfarviridae [33]. ASFV is also regarded as the only DNA virus that can be classified as an ARBO (arthropod-borne) virus due to the fact that it infects soft ticks of the genus Ornithodoros [2]. In pigs, ASFV predominantly replicates in monocytes and macrophages, although several other cell types can be infected at the final stage of disease. Infection of macrophages with several different ASFV strains leads to an increased secretory activation of proinflammatory cytokines [18]. It has been suggested that the elevated levels of cytokines such as TNF-a and IL-1b play a major role in the pathogenesis of ASF due to the proinflammatory, proapoptotic and procoagulant profile of these cytokines [18]. Thus, studies about proinflammatory cytokines during the course of infection are likely to be relevant in understanding this devastating disease. The ASFV isolates currently circulating in the TransCaucasian countries and Eastern Europe are highly virulent and grouped within genotype II [27]. Genomic analysis of
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ASFV genotype II has shown the presence of additional open reading frames, as well as divergence in proteins with immunomodulatory functions [9]. Furthermore, although the infection of pigs with ASFV genotype II leads to the clinical picture described above [21], our recent study demonstrated that ASFV genotype II causes pathological changes that differ from those caused by other highly virulent ASFV isolates [37]. In particular, it causes a decrease in the number of platelets earlier than other highly virulent strains. In contrast to other strains, ASFV genotype II infection induces a slight shortening of an activated partial thromboplastin time and no significant changes in prothrombin time and thrombin time (except in the last days of infection). These findings point to the differences between highly virulent ASFV strains and underline the importance of ASFV genotype II studies. The aim of this study was to investigate the effect of ASFV genotype II on the host immune system. Taking into consideration the role of proinflammatory cytokines, both in the immune response and in ASF pathogenesis, we measured the serum levels of TNF-a, IL-1b, IL-6, and IL8, using enzyme-linked immunosorbent assay (ELISA). In this study, we also investigated the changes in the cellular constitution of the lymphoid organs that are most commonly affected in ASF, especially the lymph nodes, spleen and bone marrow.
Materials and methods Viral stock and animal experiment The virus (Arm07) used in this study was isolated in 2007 from the spleen of a pig infected with ASFV. It belongs to genotype II, which is distributed in the Trans-Caucasian countries [27]. In this study, fifteen pigs (n = 15) of the same age (3 months old) and weight (30-32 kg) were used for infection (n = 12) and as controls (n = 3). The animals were housed in separate stables, where they had access to a commercial feed twice per day and to clear water at all times. Pigs were infected by intramuscular injection with 104 50 % hemadsorbing doses (HAD50)/ml. Nine (n = 9) pigs were used for tissue sampling and cell analysis, whereas three other pigs (n = 3) were used for blood collection and cytokine experiments. The viral titer in sera was determined by hemadsorption microtest as described previously and expressed as log10 HAD50/ml. [8, 12]. The animal experiments were approved by the Institutional Review Board/Independent Ethics Committee of the Institute of Molecular Biology of NAS RA (reference number IRB00004079).
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Tissue imprints and cell analysis Pigs were tranquilized with azaperone and euthanized with a lethal dose of sodium thiopental. Pigs were euthanized at 3, 5, 7 days postinfection (dpi) in batches of three. Fresh spleen and lymph nodes (from the abdominal cavity) were cut with a sharp blade through the hilum, and tissue imprints were made by gently touching the freshly cut surface of the tissue with a clean glass microscope slide. For bone marrow, the needle was advanced in the femoral bone marrow cavity with a twisting motion and rotated to obtain a solid piece of bone marrow, after which the imprints were made. The remaining pigs (control group) were euthanized at 7 dpi. For cell analysis, slides were fixed in pure methanol and stained with modified Giemsa solution (azure B/azure II, eosin and methylene blue) according to the manufacturer’s protocol (Sigma-Aldrich, Germany). Cells were analyzed and counted in 100 randomly selected fields (0.01 mm2) using a light microscope at 12509 magnification. In total, at least 3000 cells were analyzed and classified at each time point of infection. The microscopic evaluation of cells based on morphologic characteristics was done as described previously [20]. Tissue samples Samples from lymph nodes and spleen were fixed in 10 % buffered formalin solution (pH 7.2) for 24 hours. After fixation, the samples were dehydrated through a graded series of alcohols, washed with xylol and embedded in paraffin wax by a routine technique for light microscopy. For structural analysis, wax-embedded samples were cut (Microm HM 355, 5 lm) and stained with hematoxylin and eosin according to the manufacturer’s protocol (SigmaAldrich, Germany). The histological examination was carried out using a light microscope. Blood collection and ELISA From 1 to 7 dpi, blood samples were collected from the ophthalmic venous sinus as described previously [31]. Preinoculation (0 dpi) blood samples were taken to obtain control values. For the detection of serum levels of TNF-a, IL-1b, IL-6, and IL-8, commercial ELISA kits (Quantikine, R&D systems, USA) were used. The levels of cytokines were measured using a colorimetric reader (Stat Fax 303 Plus) and calculated according to the cytokine standard curve supplied in the kits. All samples were tested in duplicate according to manufacturers’ instructions. Pigs (n = 3) were euthanized on day 7 postinfection.
Pathology of African swine fever virus infection
Statistics Statistical tests were performed using the SPSS version 17.0 software package (SPSS Inc., Chicago, IL, USA). Data on cell analysis and cytokines were evaluated by Student’s t-test. Differences between control and infection were considered significant at the P \ 0.05 level.
Results Experimental infection Upon infection with ASFV genotype II, the first clinical signs were observed at 2-3 dpi, when all animals demonstrated fever (temperature higher than 40 °C), depression and loss of appetite. As shown in Figure 1, the average body temperature of infected animals rose greatly on day 2 (42.5 °C) and remained above 40 °C until the end of the experiment. In addition, difficulties in breathing, accelerated respiratory and pulse rates, oculonasal discharge, and reddening of the skin were seen from 3 dpi. Bloody diarrhea was observed in only three pigs. Ataxia, severe depression and lethargy occurred at 5-6 dpi, and therefore all animals were sacrificed at 7 dpi. Viremia in pigs was detected from 1 dpi (Fig. 2). The ASFV titer reached its maximum at 5 dpi (5.5 log HAD50/ml) and remained high until the end of the experiment. High titers of ASFV (5.05.5 log HAD50/ml) were observed in all pigs. Histopathological changes in spleen and lymph nodes The main lesions of lymph nodes and spleen were the depletion of cells and infiltration of red blood cells. In lymph nodes, red blood cell infiltration in parenchyma and a decrease in cell number were detected in all samples at 3,
Fig. 1 Body temperature changes after challenge with ASFV genotype II. Each point represents the mean (±SD) for all pigs on the indicated day
Fig. 2 Viral titer in pigs, expressed in log HAD50, in primary leukocytes incubated with blood from infected animals. Each point represents the mean (±SD) for all pigs on the indicated day
5 and 7 dpi (Fig. 3A and B). In spleen, microscopic lesions included red blood cell infiltration, severe depletion of follicles (reduction in size), and accumulation of hemosiderin at the sites of hemorrhage (Fig. 3C and D). No pathological changes were detected in the control group. Changes in immune cell populations Relative changes in cell populations of bone marrow, spleen and lymph nodes are summarized in Tables 1, 2 and 3, respectively. The cell population in bone marrow of uninfected animals was mainly represented by nucleated erythroid cells as well as lymphocytes and band neutrophils constituting about 60 % of the total cells (Table 1). Upon infection, the number of lymphocytes significantly decreased and reached its minimum value (14.7 %) at 7 dpi. In contrast, increases in the number of lymphoblasts and monocytes were observed at 3 dpi (Table 1). Significant changes were also observed for monoblasts (:) and band neutrophils (;). ASFV infection caused the formation of pathological forms, mainly represented by atypical lymphocytes that had an abnormal nuclear shape and hyperdiploid DNA content. In the spleens of healthy pigs, splenic cells were mainly represented by lymphocytes (Table 2). Upon ASFV infection, a twofold reduction in the number of lymphocytes was detected in the final phase of infection (Table 2). ASFV infection resulted in a decrease in the number of monocytes at 5 and 7 dpi, whereas the number of lymphoblasts was significantly lower than in the control only on the third day of observation. An increase in the percentage of myelocytes, eosinophilic metamyelocytes and nucleated erythroid cells was observed at 5 and 7 dpi. The number of atypical lymphocytes reached 3 % of splenic cells on the final day of disease (Table 2). In lymph nodes, ASFV infection also led to a twofold decrease in the number of lymphocytes at 7 dpi (Table 3).
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hemosiderin-laden cells (arrows) in spleen at 7 dpi. Tissues (5 lm) were stained with hematoxylin and eosin. Magnifications are 250 (A, B, C) and 4009 (D)
Fig. 3 Micrographs of lymph nodes and spleen. (A) Extensive infiltration of red blood cells in parenchyma of lymph nodes at 7 dpi. (B) Cell disintegration and depletion in lymph nodes at 7 dpi. (C) Reduction in size of splenic follicles at 7 dpi. (D) Presence of
Table 1 Relative changes in the cell population of bone marrow during ASFV infection
Cell type
Percent (%) of cells Control
3 dpi
5 dpi
Monoblasts
4.2 ± 0.7
5.2 ± 1.5
4.7 ± 0.4
7.1 ± 1.2*
Monocytes
3.2 ± 0.9
6.1 ± 0.9*
4.6 ± 0.5
4.2 ± 0.4
Lymphoblasts
6.5 ± 1.0
10.6 ± 0.7*
7.9 ± 1.8
8.2 ± 1.2
Lymphocytes
20.6 ± 1.5
17.1 ± 1.2
15.3 ± 2.1**
14.7 ± 1.3**
Myelocytes
9.0 ± 1.8
8.9 ± 1.3
Band neutrophils
10.6 ± 1.3
6.5 ± 1.0**
8.3 ± 1.3
6.2 ± 0.7
10.2 ± 1.6
7.9 ± 1.0
Segmented neutrophils
4.1 ± 1.4
3.4 ± 0.5
4.1 ± 0.5
3.8 ± 0.5
Eosinophils
8.5 ± 1.1
8.2 ± 1.2
6.4 ± 0.8
7.9 ± 1.0
Basophils
1.4 ± 0.6
2.7 ± 0.7
1.4 ± 0.3
2.4 ± 0.5
Nucleated erythroid cells
31.9 ± 3.8
27.5 ± 3.6
32.3 ± 4.1
32.1 ± 3.5
Pathological forms
-
3.8 ± 0.6*
4.8 ± 0.7*
Results are expressed as mean (n = 3 at each time point of infection) ± SD * Significant increase compared with control (p \ 0.05-p \ 0.001) ** Significant decrease compared with control (p \ 0.05-p \ 0.001)
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7 dpi
5.5 ± 1.0*
Pathology of African swine fever virus infection Table 2 Relative changes in cell populations in spleen during ASFV infection
Cell type
Percent (%) of cells Control
3 dpi
5 dpi
7 dpi
Monoblasts
3.1 ± 0.9
1.2 ± 0.6
-
2.4 ± 1.0
Monocytes
7.0 ± 1.2
4.5 ± 1.7
2.4 ± 0.9**
2.2 ± 0.3**
Lymphoblasts
5.7 ± 1.3
1.6 ± 0.9**
4.9 ± 1.1
Lymphocytes
60.4 ± 6.3
57.5 ± 11.0
33.4 ± 7.2**
28.5 ± 4.9**
4.4 ± 1.6 12.5 ± 2.6*
Myelocytes
1.0 ± 0.5
4.7 ± 2.8
6.8 ± 1.5*
Metamyelocytes
3.1 ± 0.8
2.6 ± 1.9
4.4 ± 1.7
4.8 ± 1.6
Band neutrophils
4.3 ± 0.7
4.5 ± 0.2
3.2 ± 0.7
7.1 ± 1.8
Segmented neutrophils
4.0 ± 0.7
3.8 ± 2.5
2.3 ± 0.8
6.5 ± 2.2
Eosinophilic metamyelocytes
1.4 ± 0.3
2.2 ± 1.4
5.7 ± 1.4*
4.9 ± 0.7*
Eosinophils
3.3 ± 1.0
10.5 ± 2.9*
17.8 ± 6.3*
6.1 ± 2.8
Basophils Nucleated erythroid cells
1.2 ± 0.6 5.5 ± 1.6
5.6 ± 1.3
1.5 ± 0.4 15.1 ± 2.4*
1.1 ± 0.3 16.5 ± 2.8*
Atypical lymphocytes
-
1.3 ± 0.3*
2.5 ± 0.8*
3.0 ± 1.0*
Results are expressed as mean (n = 3 at each time point of infection) ± SD * Significant increase compared with control (p \ 0.05-p \ 0.001) ** Significant decrease compared with control (p \ 0.05-p \ 0.001)
Table 3 Relative changes in cell populations in lymph nodes during ASFV infection
Cell types
Percent (%) of cells Control
3 dpi
5 dpi
7 dpi
Monoblasts
1.0 ± 0.2
-
1.1 ± 0.7
0.2 ± 0.1**
Monocytes
1.1 ± 0.2
0.2 ± 0.1**
2.3 ± 1.0
5.0 ± 1.3*
Lymphoblasts
1.9 ± 0.4
0.5 ± 0.1**
1.0 ± 0.6
1.0 ± 0.7
Lymphocytes
83.0 ± 5.1
90.1 ± 5.8
67.0 ± 6.3**
42.6 ± 5.9**
Myelocytes
4.3 ± 1.1
3.7 ± 0.7
3.4 ± 0.6
18.5 ± 3.4*
Metamyelocytes Band neutrophils
2.5 ± 1.0 1.2 ± 0.5
0.5 ± 0.2** 1.5 ± 0.8
2.6 ± 1.0 3.4 ± 1.4
3.7 ± 1.5 3.9 ± 2.6
Segmented neutrophils
0.8 ± 0.4
1.0 ± 0.2
1.0 ± 0.5
1.2 ± 0.8
Eosinophilic metamyelocytes
0.7 ± 0.4
0.5 ± 0.2
4.3 ± 1.2*
0.4 ± 0.3
Eosinophils
1.5 ± 0.7
1.0 ± 0.3
3.1 ± 0.9
1.6 ± 1.1
Basophils
0.6 ± 0.1
-
Nucleated erythroid cells
1.4 ± 0.1
0.7 ± 0.1**
10.1 ± 2.3*
0.4 ± 0.1
20.7 ± 3.4*
0.2 ± 0.1**
Atypical lymphocytes
-
0.3 ± 0.1*
0.3 ± 0.1*
1.0 ± 0.5*
Results are expressed as mean (n = 3 at each time point of infection) ± SD * Significant increase compared with control (p \ 0.05-p \ 0.001) ** Significant decrease compared with control (p \ 0.05-p \ 0.001)
However, in contrast to bone marrow and spleen, the number of monocytes increased to 5 % of the total cells at 7 dpi. Although no monoblasts were detected at 3 dpi, their number recovered at 5 dpi and became significantly less than in the control at 7 dpi. By the last day of infection, the number of myelocytes increased sharply and reached 18.5 % of the total cells. Other cells showed no significant changes, except nucleated erythroid cells (:) and eosinophilic metamyelocytes (: at 5 dpi). A small number of atypical lymphocytes arose and reached 1 % of the total
cells on the final day of observation (Table 3). In all tissues, the absolute number of destroyed cells increased continuously throughout the course of infection (data not shown). Levels of serum cytokines The concentrations of TNF-a, IL-1b, IL-6 and IL-8 in serum collected from ASFV-infected pigs from 0 to 7 dpi are shown in Figure 4. Upon infection, the levels
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Fig. 4 Concentrations of TNF-a (A), IL-1b (B), IL-6 (C) and IL-8 (D) in serum from ASFV-infected pigs quantified by ELISA. The cytokine levels are expressed as pg/ml. Each point represents the mean (±SD) for three (n = 3) pigs
of serum cytokines started to rise and reached a peak on the last day of experiment. No significant differences were found between controls and infected animals with regard to TNF-a level from 1 to 3 dpi (Fig. 4 A). At 4 dpi, the concentration of TNF-a was significantly higher (p \ 0.01) than in the controls and the maximum value (136.2 pg/ml) was recorded at 7 dpi. A significant elevation of IL-1b (p \ 0.05) was observed from 3 dpi and became about 5 times higher than in control on day 7 postinfection (Fig. 4 B). For IL-6, the first significant differences (p \ 0.05) were observed at 4 dpi, when the level of this cytokine reached 78.2 pg/ml (Fig. 4 C). At 7 dpi, the serum level of IL-6 (180 pg/ml) was about 10 times higher than in the control. A significantly increased level of IL-8 (p \ 0.05) was detected from day 5 (100 pg/ml) onwards (Fig. 4D).
Discussion A genomic analysis of African swine fever virus genotype II currently circulating in the Trans-Caucasian countries and Eastern Europe has revealed variations in the number of members of multigene families that may correlate with pathogenesis [9]. On the other hand, the absence of effective vaccines and antiviral agents against ASFV highlights the importance of studying the changes in the
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immune system caused by this devastating virus [10]. Thus, to determine the effect of ASFV genotype II on the host immune system, we investigated the serum concentration of proinflammatory cytokines and the cellular constitution of the lymphoid organs that are constantly targeted by ASFV. Go´mez-Villamandos et al. [16] studied the structural and ultrastructural changes of bone marrow cell populations in pigs infected with a highly virulent ASFV strain and detected severe lymphopenia from day 3 postinfection. This result is in accordance with data from our study. Go´mez-Villamandos et al. [16] also demonstrated an increased number of immature neutrophils in the bone marrow of infected animals. Interestingly, in our study, neutrophils remained unchanged (except at 3 dpi) by ASFV genotype II infection. Thus, the lack of an increase in the number of neutrophils in response to infection may point to the destruction of neutrophils or the release of immature cells from the bone marrow into the bloodstream. Our previous study showed that ASFV genotype II infection induces the appearance of metamyelocytes in the blood of infected animals, indicating the release of immature cells from the bone marrow [20]. However, the destruction of neutrophils as the second scenario cannot be completely refuted due to the fact that ASFV replication centers were previously observed within the cytoplasm of some immature and mature neutrophils [5].
Pathology of African swine fever virus infection
The spleen is among the organs that are most affected during ASFV infection [18]. Acute ASF leads to a massive accumulation of red blood cells within splenic cords [6, 15]. Moreover, lymphoid cell depletion with lymphocyte necrosis and a loss of cell junctions was observed by Carrasco et al. [3, 6]. These observations are in line with ours, showing an extensive infiltration of red blood cells in parenchyma and a decrease in the number of lymphocytes. Studies of splenic tissues also showed that red blood cell infiltration may lead to the accumulation of hemosiderin at the sites of hemorrhages. Hemosiderin is usually found in macrophages in situations following hemorrhage, suggesting that its formation may be related to phagocytosis of red blood cells and hemoglobin [35]. In contrast to lymphocytes, a significant increase in the number of splenic myelocytes and eosinophilic metamyelocytes was observed. It may be suspected that these immature cells are derived from the bloodstream and bone marrow, pointing to impaired hematopoiesis during acute ASF [21]. The lymph nodes undergo dramatic changes in structure and cellular composition in ASFV-infected pigs. It has been shown that the most frequent lesion in the lymph nodes is hemorrhage [7, 11]. Furthermore, intense lymphoid cell death by apoptosis has been observed in pigs infected with highly virulent ASFV strains [4, 26, 32]. In contrast to highly virulent strains, low levels of apoptosis in the lymph nodes have been detected in pigs infected with moderately virulent strains, indicating that the degree of apoptosis is directly related to the viral virulence [25]. Our study demonstrated that experimental infection of pigs with ASFV genotype II led to a significant decrease in the number of lymphocytes on the final day of the experiment, which is in line with other studies. Thus, this and other work provide evidence that lymphoid cell depletion is one of the most important characteristics of ASFV infection. In addition, our studies demonstrate that acute ASFV infection is accompanied by the formation of atypical lymphocytes, both in vitro and in vivo [20–22]. Viruses may affect lymphocytes directly or indirectly, and a number of mechanisms exist to explain the apoptosis of lymphoid cells [23, 24, 30, 34]. However, the factors that contribute to the severe lymphopenia observed in ASF are still poorly investigated. Although Wardley et al. [36] showed that purified lymphocytes are infected by virulent ASFV strains, a direct effect on lymphocytes can be ruled out as a possible cause of apoptosis due to the fact that only a small number of mature ASFV particles are detected in lymphocytes. On the other hand, a large body of evidence suggests a role of proinflammatory cytokines secreted by activated or infected macrophages, thereby highlighting the indirect effect of ASFV on lymphocytes. In particular, it has been shown that TNF-a secreted by ASFV-infected macrophages induces the apoptotic process in porcine
lymphocytes [14]. Furthermore, an increase in proinflammatory cytokine (TNF-a, IL-1a, IL-1b and IL-6) expression in lymphoid organs accompanied by increased apoptosis of lymphocytes was detected by Salguero et al. [28, 29]. These findings support the assumption that proinflammatory cytokines can induce lymphocyte apoptosis in acute ASF. The results of this study demonstrated that pigs infected with ASFV genotype II have increased levels of serum proinflammatory cytokines. This may explain not only the lymphocyte depletion observed in the lymphoid organs but also the formation of atypical lymphocytes that have hyperdiploid DNA content [20, 21]. It has been shown in a recent study that IL-8 increases the synthesis of DNA in human epithelial cells [13]. Therefore, further studies are required to determine the role of proinflammatory cytokines in the formation of atypical lymphocytes. In conclusion, this study demonstrates that ASFV genotype II causes a dramatic decrease in the number of lymphocytes in the immune organs. Furthermore, the accumulation of immature immune cells in these organs shows that acute ASF is accompanied by impaired hematopoiesis. The elevated levels of TNF-a, IL-1b, IL-6 and IL-8 are likely to be responsible for the depletion of lymphoid cells. However, whether these cytokines also contribute to the formation of atypical lymphocytes described in our studies remains unclear. Finally, this study indicates that pigs infected with a highly virulent ASFV genotype II strain undergo severe immunopathological changes, and further studies will be needed to for understanding the pathogenesis of ASFV. Acknowledgments This work was financially supported by the Institute of Molecular Biology of NAS RA. The authors thank laboratory members, as well as all reviewers for their time spent on reviewing the manuscript, and for the helpful comments and suggestions. Conflict of interest
The authors declare no conflict of interest.
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porcine bone marrow during infection of African swine fever virus. In Vitro Cell Dev Biol Anim 47:200–204 Karalyan Z, Zakaryan H, Arzumanyan H, Sargsyan K, Voskanyan H, Hakobyan L, Abroyan L, Avetisyan A, Karalova E (2012) Pathology of porcine peripheral white blood cells during infection with African swine fever virus. BMC Vet Res 8:18 Karalyan Z, Zakaryan H, Sargsyan Kh, Voskanyan H, Arzumanyan H, Avagyan H, Karalova E (2012) Interferon status and white blood cells during infection with African swine fever virus in vivo. Vet Immunol Immunopathol 145:551–555 Lafon M (2011) Evasive strategies in rabies virus infection. Adv Virus Res 79:33–53 Mohamadzadeh M (2009) Potential factors induced by filoviruses that lead to immune supression. Curr Mol Med 9:174–185 Oura CA, Powell PP, Parkhouse RM (1998) African swine fever: a disease characterized by apoptosis. J Gen Virol 79:1427–1438 Ramiro-Iba´n˜ez F, Ortega A, Brun A, Escribano JM, Alonso C (1996) Apoptosis: a mechanism of cell killing and lymphoid organ impairment during acute African swine fever virus infection. J Gen Virol 77:2209–2219 Rowlands RJ, Michaud V, Heath L, Hutchings G, Oura C, Vosloo W, Dwarka R, Onashvili T, Albina E, Dixon LK (2008) African swine fever virus isolate, Georgia, 2007. Emerg Infect Dis 14:1870–1874 Salguero FJ, Ruiz-Villamor E, Bautista MJ, Sa´nchez-Cordo´n PJ, Carrasco L, Go´mez-Villamandos JC (2002) Changes in macrophages in spleen and lymph nodes during acute African swine fever: expression of cytokines. Vet Immunol Immunopathol 90:11–22 Salguero FJ, Sa´nchez-Cordo´n PJ, Nu´n˜ez A, Ferna´ndez de Marco M, Go´mez-Villamandos JC (2005) Proinflammatory cytokines induce lymphocyte apoptosis in acute African swine fever infection. J Comp Pathol 132:289–302 Schat KA (2009) Chicken anemia virus. Curr Top Microbiol Immunol 331:151–183 Stier H, Leucht W (1980) Blood sampling from the venous ophthalmic sinus of miniature swine. Z Versuchstierkd 22:161–164 Takamatsu H, Denyer MS, Oura C, Childerstone A, Andersen JK, Pullen L, Parkhouse RM (1999) African swine fever virus: a B cell-mitogenic virus in vivo and in vitro. J Gen Virol 80:1453–1461 Takamatsu H, Martins C, Escribano JM, Alonso C, Dixon LK, Salas ML, Revilla Y (2011) Asfarviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus taxonomy. Ninth report of the ICTV. Elsevier, Oxford, pp 153–162 Varbanov M, Espert L, Biard-Piechaczyk M (2006) Mechanisms of CD4 T-cell depletion triggered by HIV-1 viral proteins. AIDS Rev 8:221–236 Wang Y, Juan LV, Ma X, Wang D, Ma H, Chang Y, Nie G, Jia L, Duan X, Liang XJ (2010) Specific hemosiderin deposition in spleen induced by a low dose of cisplatin: altered iron metabolism and its implication as an acute hemosiderin formation model. Curr Drug Metab 11:507–515 Wardley RC, Wilkinson PJ, Hamilton F (1977) African swine fever virus replication in porcine lymphocytes. J Gen Virol 37:425–427 Zakaryan H, Karalova E, Voskanyan H, Ter-Pogossyan Z, Nersisyan N, Hakobyan A, Saroyan D, Karalyan Z (2014) Evaluation of hemostaseological status of pigs experimentally infected with African swine fever virus. Vet Microbiol 174:223–228
ORIGINAL ARTICLE
A study of lymphoid organs and serum proinflammatory cytokines in pigs infected with African swine fever virus genotype II Hovakim Zakaryan1 • Victorya Cholakyans1 • Lusine Simonyan1 • Alla Misakyan1 Elena Karalova1 • Andranik Chavushyan2 • Zaven Karalyan1
•
Received: 5 December 2014 / Accepted: 15 March 2015 Ó Springer-Verlag Wien 2015
Abstract African swine fever virus (ASFV), the causative agent of one of the most important viral diseases of domestic pigs for which no vaccine is available, causes immune system disorders in infected animals. In this study, the serum levels of proinflammatory cytokines, as well as the histological and cellular constitution of lymphoid organs of pigs infected with ASFV genotype II were investigated. The results showed a high degree of lymphocyte depletion in the lymphoid organs, particularly in the spleen and lymph nodes, where ASFV infection led to a twofold decrease in the number of lymphocytes on the final day of infection. Additionally, ASFV-infected pigs had atypical forms of lymphocytes found in all lymphoid organs. In contrast to lymphocytes, the number of immature immune cells, particularly myelocytes, increased dramatically and reached a maximum on day 7 postinfection. The serum levels of TNF-a, IL-1b, IL-6, and IL-8 were evaluated. Proinflammatory cytokines showed increased levels after ASFV infection, with peak values at 7 days postinfection, and this highlights their role in the pathogenesis of ASFV. In conclusion, this study showed that ASFV genotype II, like other highly virulent strains, causes severe pathological changes in the immune system of pigs.
& Zaven Karalyan [email protected] 1
Laboratory of Cell Biology and Virology, Institute of Molecular Biology of NAS RA, 0014 Yerevan, Armenia
2
Laboratory of Human Genomics and Immunomics, Institute of Molecular Biology of NAS RA, 0014 Yerevan, Armenia
Introduction African swine fever (ASF) is a highly contagious disease affecting domestic pigs and wild suids. In acute forms of this disease, symptoms in domestic pigs include fever, vascular changes (particularly bleeding), cyanosis of the skin, abdominal pain, and diarrhoea, which may become bloody prior to death [1, 17, 19]. These clinical signs are accompanied by severe thrombocytopenia and lymphopenia. Pigs usually die in less than 10 days. Due to its high mortality rate, which approaches 100 %, and the lack of vaccines, ASF is one of the most important viral diseases of domestic pigs. African swine fever virus (ASFV), the causative agent of ASF, is a large, enveloped double-stranded DNA virus. It is the only member of the genus Asfivirus within the family Asfarviridae [33]. ASFV is also regarded as the only DNA virus that can be classified as an ARBO (arthropod-borne) virus due to the fact that it infects soft ticks of the genus Ornithodoros [2]. In pigs, ASFV predominantly replicates in monocytes and macrophages, although several other cell types can be infected at the final stage of disease. Infection of macrophages with several different ASFV strains leads to an increased secretory activation of proinflammatory cytokines [18]. It has been suggested that the elevated levels of cytokines such as TNF-a and IL-1b play a major role in the pathogenesis of ASF due to the proinflammatory, proapoptotic and procoagulant profile of these cytokines [18]. Thus, studies about proinflammatory cytokines during the course of infection are likely to be relevant in understanding this devastating disease. The ASFV isolates currently circulating in the TransCaucasian countries and Eastern Europe are highly virulent and grouped within genotype II [27]. Genomic analysis of
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ASFV genotype II has shown the presence of additional open reading frames, as well as divergence in proteins with immunomodulatory functions [9]. Furthermore, although the infection of pigs with ASFV genotype II leads to the clinical picture described above [21], our recent study demonstrated that ASFV genotype II causes pathological changes that differ from those caused by other highly virulent ASFV isolates [37]. In particular, it causes a decrease in the number of platelets earlier than other highly virulent strains. In contrast to other strains, ASFV genotype II infection induces a slight shortening of an activated partial thromboplastin time and no significant changes in prothrombin time and thrombin time (except in the last days of infection). These findings point to the differences between highly virulent ASFV strains and underline the importance of ASFV genotype II studies. The aim of this study was to investigate the effect of ASFV genotype II on the host immune system. Taking into consideration the role of proinflammatory cytokines, both in the immune response and in ASF pathogenesis, we measured the serum levels of TNF-a, IL-1b, IL-6, and IL8, using enzyme-linked immunosorbent assay (ELISA). In this study, we also investigated the changes in the cellular constitution of the lymphoid organs that are most commonly affected in ASF, especially the lymph nodes, spleen and bone marrow.
Materials and methods Viral stock and animal experiment The virus (Arm07) used in this study was isolated in 2007 from the spleen of a pig infected with ASFV. It belongs to genotype II, which is distributed in the Trans-Caucasian countries [27]. In this study, fifteen pigs (n = 15) of the same age (3 months old) and weight (30-32 kg) were used for infection (n = 12) and as controls (n = 3). The animals were housed in separate stables, where they had access to a commercial feed twice per day and to clear water at all times. Pigs were infected by intramuscular injection with 104 50 % hemadsorbing doses (HAD50)/ml. Nine (n = 9) pigs were used for tissue sampling and cell analysis, whereas three other pigs (n = 3) were used for blood collection and cytokine experiments. The viral titer in sera was determined by hemadsorption microtest as described previously and expressed as log10 HAD50/ml. [8, 12]. The animal experiments were approved by the Institutional Review Board/Independent Ethics Committee of the Institute of Molecular Biology of NAS RA (reference number IRB00004079).
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Tissue imprints and cell analysis Pigs were tranquilized with azaperone and euthanized with a lethal dose of sodium thiopental. Pigs were euthanized at 3, 5, 7 days postinfection (dpi) in batches of three. Fresh spleen and lymph nodes (from the abdominal cavity) were cut with a sharp blade through the hilum, and tissue imprints were made by gently touching the freshly cut surface of the tissue with a clean glass microscope slide. For bone marrow, the needle was advanced in the femoral bone marrow cavity with a twisting motion and rotated to obtain a solid piece of bone marrow, after which the imprints were made. The remaining pigs (control group) were euthanized at 7 dpi. For cell analysis, slides were fixed in pure methanol and stained with modified Giemsa solution (azure B/azure II, eosin and methylene blue) according to the manufacturer’s protocol (Sigma-Aldrich, Germany). Cells were analyzed and counted in 100 randomly selected fields (0.01 mm2) using a light microscope at 12509 magnification. In total, at least 3000 cells were analyzed and classified at each time point of infection. The microscopic evaluation of cells based on morphologic characteristics was done as described previously [20]. Tissue samples Samples from lymph nodes and spleen were fixed in 10 % buffered formalin solution (pH 7.2) for 24 hours. After fixation, the samples were dehydrated through a graded series of alcohols, washed with xylol and embedded in paraffin wax by a routine technique for light microscopy. For structural analysis, wax-embedded samples were cut (Microm HM 355, 5 lm) and stained with hematoxylin and eosin according to the manufacturer’s protocol (SigmaAldrich, Germany). The histological examination was carried out using a light microscope. Blood collection and ELISA From 1 to 7 dpi, blood samples were collected from the ophthalmic venous sinus as described previously [31]. Preinoculation (0 dpi) blood samples were taken to obtain control values. For the detection of serum levels of TNF-a, IL-1b, IL-6, and IL-8, commercial ELISA kits (Quantikine, R&D systems, USA) were used. The levels of cytokines were measured using a colorimetric reader (Stat Fax 303 Plus) and calculated according to the cytokine standard curve supplied in the kits. All samples were tested in duplicate according to manufacturers’ instructions. Pigs (n = 3) were euthanized on day 7 postinfection.
Pathology of African swine fever virus infection
Statistics Statistical tests were performed using the SPSS version 17.0 software package (SPSS Inc., Chicago, IL, USA). Data on cell analysis and cytokines were evaluated by Student’s t-test. Differences between control and infection were considered significant at the P \ 0.05 level.
Results Experimental infection Upon infection with ASFV genotype II, the first clinical signs were observed at 2-3 dpi, when all animals demonstrated fever (temperature higher than 40 °C), depression and loss of appetite. As shown in Figure 1, the average body temperature of infected animals rose greatly on day 2 (42.5 °C) and remained above 40 °C until the end of the experiment. In addition, difficulties in breathing, accelerated respiratory and pulse rates, oculonasal discharge, and reddening of the skin were seen from 3 dpi. Bloody diarrhea was observed in only three pigs. Ataxia, severe depression and lethargy occurred at 5-6 dpi, and therefore all animals were sacrificed at 7 dpi. Viremia in pigs was detected from 1 dpi (Fig. 2). The ASFV titer reached its maximum at 5 dpi (5.5 log HAD50/ml) and remained high until the end of the experiment. High titers of ASFV (5.05.5 log HAD50/ml) were observed in all pigs. Histopathological changes in spleen and lymph nodes The main lesions of lymph nodes and spleen were the depletion of cells and infiltration of red blood cells. In lymph nodes, red blood cell infiltration in parenchyma and a decrease in cell number were detected in all samples at 3,
Fig. 1 Body temperature changes after challenge with ASFV genotype II. Each point represents the mean (±SD) for all pigs on the indicated day
Fig. 2 Viral titer in pigs, expressed in log HAD50, in primary leukocytes incubated with blood from infected animals. Each point represents the mean (±SD) for all pigs on the indicated day
5 and 7 dpi (Fig. 3A and B). In spleen, microscopic lesions included red blood cell infiltration, severe depletion of follicles (reduction in size), and accumulation of hemosiderin at the sites of hemorrhage (Fig. 3C and D). No pathological changes were detected in the control group. Changes in immune cell populations Relative changes in cell populations of bone marrow, spleen and lymph nodes are summarized in Tables 1, 2 and 3, respectively. The cell population in bone marrow of uninfected animals was mainly represented by nucleated erythroid cells as well as lymphocytes and band neutrophils constituting about 60 % of the total cells (Table 1). Upon infection, the number of lymphocytes significantly decreased and reached its minimum value (14.7 %) at 7 dpi. In contrast, increases in the number of lymphoblasts and monocytes were observed at 3 dpi (Table 1). Significant changes were also observed for monoblasts (:) and band neutrophils (;). ASFV infection caused the formation of pathological forms, mainly represented by atypical lymphocytes that had an abnormal nuclear shape and hyperdiploid DNA content. In the spleens of healthy pigs, splenic cells were mainly represented by lymphocytes (Table 2). Upon ASFV infection, a twofold reduction in the number of lymphocytes was detected in the final phase of infection (Table 2). ASFV infection resulted in a decrease in the number of monocytes at 5 and 7 dpi, whereas the number of lymphoblasts was significantly lower than in the control only on the third day of observation. An increase in the percentage of myelocytes, eosinophilic metamyelocytes and nucleated erythroid cells was observed at 5 and 7 dpi. The number of atypical lymphocytes reached 3 % of splenic cells on the final day of disease (Table 2). In lymph nodes, ASFV infection also led to a twofold decrease in the number of lymphocytes at 7 dpi (Table 3).
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hemosiderin-laden cells (arrows) in spleen at 7 dpi. Tissues (5 lm) were stained with hematoxylin and eosin. Magnifications are 250 (A, B, C) and 4009 (D)
Fig. 3 Micrographs of lymph nodes and spleen. (A) Extensive infiltration of red blood cells in parenchyma of lymph nodes at 7 dpi. (B) Cell disintegration and depletion in lymph nodes at 7 dpi. (C) Reduction in size of splenic follicles at 7 dpi. (D) Presence of
Table 1 Relative changes in the cell population of bone marrow during ASFV infection
Cell type
Percent (%) of cells Control
3 dpi
5 dpi
Monoblasts
4.2 ± 0.7
5.2 ± 1.5
4.7 ± 0.4
7.1 ± 1.2*
Monocytes
3.2 ± 0.9
6.1 ± 0.9*
4.6 ± 0.5
4.2 ± 0.4
Lymphoblasts
6.5 ± 1.0
10.6 ± 0.7*
7.9 ± 1.8
8.2 ± 1.2
Lymphocytes
20.6 ± 1.5
17.1 ± 1.2
15.3 ± 2.1**
14.7 ± 1.3**
Myelocytes
9.0 ± 1.8
8.9 ± 1.3
Band neutrophils
10.6 ± 1.3
6.5 ± 1.0**
8.3 ± 1.3
6.2 ± 0.7
10.2 ± 1.6
7.9 ± 1.0
Segmented neutrophils
4.1 ± 1.4
3.4 ± 0.5
4.1 ± 0.5
3.8 ± 0.5
Eosinophils
8.5 ± 1.1
8.2 ± 1.2
6.4 ± 0.8
7.9 ± 1.0
Basophils
1.4 ± 0.6
2.7 ± 0.7
1.4 ± 0.3
2.4 ± 0.5
Nucleated erythroid cells
31.9 ± 3.8
27.5 ± 3.6
32.3 ± 4.1
32.1 ± 3.5
Pathological forms
-
3.8 ± 0.6*
4.8 ± 0.7*
Results are expressed as mean (n = 3 at each time point of infection) ± SD * Significant increase compared with control (p \ 0.05-p \ 0.001) ** Significant decrease compared with control (p \ 0.05-p \ 0.001)
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7 dpi
5.5 ± 1.0*
Pathology of African swine fever virus infection Table 2 Relative changes in cell populations in spleen during ASFV infection
Cell type
Percent (%) of cells Control
3 dpi
5 dpi
7 dpi
Monoblasts
3.1 ± 0.9
1.2 ± 0.6
-
2.4 ± 1.0
Monocytes
7.0 ± 1.2
4.5 ± 1.7
2.4 ± 0.9**
2.2 ± 0.3**
Lymphoblasts
5.7 ± 1.3
1.6 ± 0.9**
4.9 ± 1.1
Lymphocytes
60.4 ± 6.3
57.5 ± 11.0
33.4 ± 7.2**
28.5 ± 4.9**
4.4 ± 1.6 12.5 ± 2.6*
Myelocytes
1.0 ± 0.5
4.7 ± 2.8
6.8 ± 1.5*
Metamyelocytes
3.1 ± 0.8
2.6 ± 1.9
4.4 ± 1.7
4.8 ± 1.6
Band neutrophils
4.3 ± 0.7
4.5 ± 0.2
3.2 ± 0.7
7.1 ± 1.8
Segmented neutrophils
4.0 ± 0.7
3.8 ± 2.5
2.3 ± 0.8
6.5 ± 2.2
Eosinophilic metamyelocytes
1.4 ± 0.3
2.2 ± 1.4
5.7 ± 1.4*
4.9 ± 0.7*
Eosinophils
3.3 ± 1.0
10.5 ± 2.9*
17.8 ± 6.3*
6.1 ± 2.8
Basophils Nucleated erythroid cells
1.2 ± 0.6 5.5 ± 1.6
5.6 ± 1.3
1.5 ± 0.4 15.1 ± 2.4*
1.1 ± 0.3 16.5 ± 2.8*
Atypical lymphocytes
-
1.3 ± 0.3*
2.5 ± 0.8*
3.0 ± 1.0*
Results are expressed as mean (n = 3 at each time point of infection) ± SD * Significant increase compared with control (p \ 0.05-p \ 0.001) ** Significant decrease compared with control (p \ 0.05-p \ 0.001)
Table 3 Relative changes in cell populations in lymph nodes during ASFV infection
Cell types
Percent (%) of cells Control
3 dpi
5 dpi
7 dpi
Monoblasts
1.0 ± 0.2
-
1.1 ± 0.7
0.2 ± 0.1**
Monocytes
1.1 ± 0.2
0.2 ± 0.1**
2.3 ± 1.0
5.0 ± 1.3*
Lymphoblasts
1.9 ± 0.4
0.5 ± 0.1**
1.0 ± 0.6
1.0 ± 0.7
Lymphocytes
83.0 ± 5.1
90.1 ± 5.8
67.0 ± 6.3**
42.6 ± 5.9**
Myelocytes
4.3 ± 1.1
3.7 ± 0.7
3.4 ± 0.6
18.5 ± 3.4*
Metamyelocytes Band neutrophils
2.5 ± 1.0 1.2 ± 0.5
0.5 ± 0.2** 1.5 ± 0.8
2.6 ± 1.0 3.4 ± 1.4
3.7 ± 1.5 3.9 ± 2.6
Segmented neutrophils
0.8 ± 0.4
1.0 ± 0.2
1.0 ± 0.5
1.2 ± 0.8
Eosinophilic metamyelocytes
0.7 ± 0.4
0.5 ± 0.2
4.3 ± 1.2*
0.4 ± 0.3
Eosinophils
1.5 ± 0.7
1.0 ± 0.3
3.1 ± 0.9
1.6 ± 1.1
Basophils
0.6 ± 0.1
-
Nucleated erythroid cells
1.4 ± 0.1
0.7 ± 0.1**
10.1 ± 2.3*
0.4 ± 0.1
20.7 ± 3.4*
0.2 ± 0.1**
Atypical lymphocytes
-
0.3 ± 0.1*
0.3 ± 0.1*
1.0 ± 0.5*
Results are expressed as mean (n = 3 at each time point of infection) ± SD * Significant increase compared with control (p \ 0.05-p \ 0.001) ** Significant decrease compared with control (p \ 0.05-p \ 0.001)
However, in contrast to bone marrow and spleen, the number of monocytes increased to 5 % of the total cells at 7 dpi. Although no monoblasts were detected at 3 dpi, their number recovered at 5 dpi and became significantly less than in the control at 7 dpi. By the last day of infection, the number of myelocytes increased sharply and reached 18.5 % of the total cells. Other cells showed no significant changes, except nucleated erythroid cells (:) and eosinophilic metamyelocytes (: at 5 dpi). A small number of atypical lymphocytes arose and reached 1 % of the total
cells on the final day of observation (Table 3). In all tissues, the absolute number of destroyed cells increased continuously throughout the course of infection (data not shown). Levels of serum cytokines The concentrations of TNF-a, IL-1b, IL-6 and IL-8 in serum collected from ASFV-infected pigs from 0 to 7 dpi are shown in Figure 4. Upon infection, the levels
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Fig. 4 Concentrations of TNF-a (A), IL-1b (B), IL-6 (C) and IL-8 (D) in serum from ASFV-infected pigs quantified by ELISA. The cytokine levels are expressed as pg/ml. Each point represents the mean (±SD) for three (n = 3) pigs
of serum cytokines started to rise and reached a peak on the last day of experiment. No significant differences were found between controls and infected animals with regard to TNF-a level from 1 to 3 dpi (Fig. 4 A). At 4 dpi, the concentration of TNF-a was significantly higher (p \ 0.01) than in the controls and the maximum value (136.2 pg/ml) was recorded at 7 dpi. A significant elevation of IL-1b (p \ 0.05) was observed from 3 dpi and became about 5 times higher than in control on day 7 postinfection (Fig. 4 B). For IL-6, the first significant differences (p \ 0.05) were observed at 4 dpi, when the level of this cytokine reached 78.2 pg/ml (Fig. 4 C). At 7 dpi, the serum level of IL-6 (180 pg/ml) was about 10 times higher than in the control. A significantly increased level of IL-8 (p \ 0.05) was detected from day 5 (100 pg/ml) onwards (Fig. 4D).
Discussion A genomic analysis of African swine fever virus genotype II currently circulating in the Trans-Caucasian countries and Eastern Europe has revealed variations in the number of members of multigene families that may correlate with pathogenesis [9]. On the other hand, the absence of effective vaccines and antiviral agents against ASFV highlights the importance of studying the changes in the
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immune system caused by this devastating virus [10]. Thus, to determine the effect of ASFV genotype II on the host immune system, we investigated the serum concentration of proinflammatory cytokines and the cellular constitution of the lymphoid organs that are constantly targeted by ASFV. Go´mez-Villamandos et al. [16] studied the structural and ultrastructural changes of bone marrow cell populations in pigs infected with a highly virulent ASFV strain and detected severe lymphopenia from day 3 postinfection. This result is in accordance with data from our study. Go´mez-Villamandos et al. [16] also demonstrated an increased number of immature neutrophils in the bone marrow of infected animals. Interestingly, in our study, neutrophils remained unchanged (except at 3 dpi) by ASFV genotype II infection. Thus, the lack of an increase in the number of neutrophils in response to infection may point to the destruction of neutrophils or the release of immature cells from the bone marrow into the bloodstream. Our previous study showed that ASFV genotype II infection induces the appearance of metamyelocytes in the blood of infected animals, indicating the release of immature cells from the bone marrow [20]. However, the destruction of neutrophils as the second scenario cannot be completely refuted due to the fact that ASFV replication centers were previously observed within the cytoplasm of some immature and mature neutrophils [5].
Pathology of African swine fever virus infection
The spleen is among the organs that are most affected during ASFV infection [18]. Acute ASF leads to a massive accumulation of red blood cells within splenic cords [6, 15]. Moreover, lymphoid cell depletion with lymphocyte necrosis and a loss of cell junctions was observed by Carrasco et al. [3, 6]. These observations are in line with ours, showing an extensive infiltration of red blood cells in parenchyma and a decrease in the number of lymphocytes. Studies of splenic tissues also showed that red blood cell infiltration may lead to the accumulation of hemosiderin at the sites of hemorrhages. Hemosiderin is usually found in macrophages in situations following hemorrhage, suggesting that its formation may be related to phagocytosis of red blood cells and hemoglobin [35]. In contrast to lymphocytes, a significant increase in the number of splenic myelocytes and eosinophilic metamyelocytes was observed. It may be suspected that these immature cells are derived from the bloodstream and bone marrow, pointing to impaired hematopoiesis during acute ASF [21]. The lymph nodes undergo dramatic changes in structure and cellular composition in ASFV-infected pigs. It has been shown that the most frequent lesion in the lymph nodes is hemorrhage [7, 11]. Furthermore, intense lymphoid cell death by apoptosis has been observed in pigs infected with highly virulent ASFV strains [4, 26, 32]. In contrast to highly virulent strains, low levels of apoptosis in the lymph nodes have been detected in pigs infected with moderately virulent strains, indicating that the degree of apoptosis is directly related to the viral virulence [25]. Our study demonstrated that experimental infection of pigs with ASFV genotype II led to a significant decrease in the number of lymphocytes on the final day of the experiment, which is in line with other studies. Thus, this and other work provide evidence that lymphoid cell depletion is one of the most important characteristics of ASFV infection. In addition, our studies demonstrate that acute ASFV infection is accompanied by the formation of atypical lymphocytes, both in vitro and in vivo [20–22]. Viruses may affect lymphocytes directly or indirectly, and a number of mechanisms exist to explain the apoptosis of lymphoid cells [23, 24, 30, 34]. However, the factors that contribute to the severe lymphopenia observed in ASF are still poorly investigated. Although Wardley et al. [36] showed that purified lymphocytes are infected by virulent ASFV strains, a direct effect on lymphocytes can be ruled out as a possible cause of apoptosis due to the fact that only a small number of mature ASFV particles are detected in lymphocytes. On the other hand, a large body of evidence suggests a role of proinflammatory cytokines secreted by activated or infected macrophages, thereby highlighting the indirect effect of ASFV on lymphocytes. In particular, it has been shown that TNF-a secreted by ASFV-infected macrophages induces the apoptotic process in porcine
lymphocytes [14]. Furthermore, an increase in proinflammatory cytokine (TNF-a, IL-1a, IL-1b and IL-6) expression in lymphoid organs accompanied by increased apoptosis of lymphocytes was detected by Salguero et al. [28, 29]. These findings support the assumption that proinflammatory cytokines can induce lymphocyte apoptosis in acute ASF. The results of this study demonstrated that pigs infected with ASFV genotype II have increased levels of serum proinflammatory cytokines. This may explain not only the lymphocyte depletion observed in the lymphoid organs but also the formation of atypical lymphocytes that have hyperdiploid DNA content [20, 21]. It has been shown in a recent study that IL-8 increases the synthesis of DNA in human epithelial cells [13]. Therefore, further studies are required to determine the role of proinflammatory cytokines in the formation of atypical lymphocytes. In conclusion, this study demonstrates that ASFV genotype II causes a dramatic decrease in the number of lymphocytes in the immune organs. Furthermore, the accumulation of immature immune cells in these organs shows that acute ASF is accompanied by impaired hematopoiesis. The elevated levels of TNF-a, IL-1b, IL-6 and IL-8 are likely to be responsible for the depletion of lymphoid cells. However, whether these cytokines also contribute to the formation of atypical lymphocytes described in our studies remains unclear. Finally, this study indicates that pigs infected with a highly virulent ASFV genotype II strain undergo severe immunopathological changes, and further studies will be needed to for understanding the pathogenesis of ASFV. Acknowledgments This work was financially supported by the Institute of Molecular Biology of NAS RA. The authors thank laboratory members, as well as all reviewers for their time spent on reviewing the manuscript, and for the helpful comments and suggestions. Conflict of interest
The authors declare no conflict of interest.
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