Since it has been shown that highly virulent ASFV strains target the mononuclear-phagocytic system, including macrophages, and destruct lymphoid tissues, it would be hypothesized that several other forms of white blood cells, such as lymphocytes, might be sensitive to ASFV infection. Indeed, it has been noted that highly virulent isolates of ASFV induce massive lymphopenia in vivo and in vitro [13
]. The factors that are responsible for lymphopenia and the precise mechanism of lymphocyte apoptosis are still unknown. However, it has been suggested that tumor necrosis factor-alpha and interleukin-1 act as cytotoxic factors and their high expression is implicated in lymphopenia [19
]. In addition to lymphopenia, we have shown that acute ASFV infection in vitro leads to the emergence of atypical lymphocytes, with abnormal nuclear shape and DNA content, indicating that ASFV may induce the formation of atypical cells in vivo [8
This paper describes the changes in population of peripheral white blood cells of infected pigs. We observed that the infection of healthy pigs with ASFV led to profound changes in blood composition [20
]. Particularly, the number of white blood cells significantly decreased from 3 dpi, coinciding with lymphopenia caused by apoptosis of lymphocytes and necrosis of lymphoid tissues [15
]. Herein we observed a band-to-segmented neutrophils ratio that became 3.0 (left shift) at the later stages of infection, indicating the emergence of immature neutrophils in blood. Some anomalies, such as impaired nuclear segmentation of mature neutrophilic granulocytes, mimic shift of neutrophils to the left [21
]. However, metamyelocytes were detected only in the blood of infected pigs, suggesting that the high number of immature neutrophils was a particular consequence of left shift and not a result of mimicry.
Other immature cells, such as lymphoblasts and monoblasts, were also observed in the blood of ASFV-infected pigs. Our data indicate that ASFV infection directly or indirectly influences on releasing of lymphoblasts and monoblasts from the bone marrow. The emergence of immature cells in peripheral blood can be attributed to activation of hematopoiesis in bone marrow. Although increased hematopoiesis might exert a positive influence on the host response to acute infection, the hematologic data obtained in this and other studies suggest that ASFV infection leads to impaired hematopoiesis [13
]. Cytometry of lymphoblasts and monoblasts revealed redundant DNA content from 3-4 dpi. This observation accords with previous studies where it was noted that some immature white blood cells had hyperdiploid DNA content [22
]. The final phase of the disease was characterized not only by hyperdiploid cells but also by lymphoblasts in which DNA content was less than in diploid cells (i.e. hypodiploid). This phenomenon might be explained by degradation of DNA due to apoptosis of lymphocytes [16
In addition to the previous study, when we detected atypical lymphocytes in primary culture of bone marrow during ASFV infection, the current study shows that atypical lymphocytes also arise as a consequence of in vivo infection. Shiftan and Mendelsohn pointed out the criteria that can be used for definition of atypical lymphocytes [26
]. One of the criteria is the ability of atypical lymphocytes to synthesize DNA, whereas non-pathologic lymphocytes are disabled to do so. Although atypical lymphocytes were observed during the entire course of infection, hyperdiploid DNA content occurred in the final phase of infection. Thus, we can hypothesize that atypical lymphocytes detected at early days of infection were transient forms that became atypical after DNA synthesis [27
]. The precise mechanism for DNA synthesis remains to be elucidated, but our working hypothesis is that ASFV directly or indirectly influences the mitotic activity of lymphocytes, leading to incomplete mitosis. This hypothesis is supported by the experiments demonstrating that other DNA viruses, such as human papillomavirus type 1, are able to induce S phase entry in cells that would otherwise be in G0 as well as arrest at G2/M [28
]. Therefore, it would be interesting to carry out a series of experiments to determine whether ASFV interferes in the cell cycle of lymphocytes or lymphoblasts.