This study revealed that Mo-DC and its progenitor cells (i.e., monocytes) could become infected with the BVDV virus; and while monocytes subsequently produced infectious virus, Mo-DC did not. Furthermore, virus production decreased with monocyte differentiation to Mo-DC and had completely ceased by120 hours of differentiation; thus, monocytes lost the ability to produce infectious virus with differentiation to Mo-DC. It was also determined that Mo-DC supported BVDV viral RNA replication but; the kinetics of viral RNA production was different between different viral strains. Additionally, viral proteins were translated in BVDV infected Mo-DC; thus, the accumulation of BVDV viral RNA and its subsequent translation into viral proteins suggest that failure to produce infectious virus may be the result of a hindrance in viral assembly, packaging, or release in Mo-DC. Further studies using electron microscopy may confirm the actual level of hindrance in infectious virus production.
A previous study that utilized a homologous pair of BVDV ncp and cp viruses (i.e., Pe515ncp and Pe515cp) revealed that Mo-DC were able to replicate and produce infectious BVDV . The difference in the ability of the Mo-DC to produce infectious virus between that study and the present study may be due to the use of different monocyte isolation and Mo-DC culturing methods, as the previous study utilized CD14+Mo-DC, while the current study examined Mo-DC that were determined to be non-adherent CD14- cells. CD14 is a cell surface marker specifically for monocytes and macrophages. It acts as a co-receptor for bacterial lipopolysaccharide (LPS) with toll-like receptor- 4 (TLR 4) . CD14 expression is reduced during differentiation of monocytes to Mo-DC [13, 14]. Likewise, in another study, bovine CD11b+Mo-DC also produced infectious BVDV; however, that study did not describe the culturing method, phenotype, or morphology of the Mo-DC used . CD11b is an adhesion molecule and complement receptor that is expressed on neutrophils, monocytes, macrophages and Mo-DCs [16, 17]. The above cell surface molecules in combination with DEC205 are routinely used to characterize the Mo-DC. DEC205 is a specific marker of Mo-DC .
A number of studies have been performed using Mo-DC and hepatitis C virus (HCV; another member of the Flaviviridae family). Similarly, both BVDV and HCV have a similar structural organization, can cause chronic infections in their respective hosts, utilize the low-density lipoprotein (LDL) receptor to enter the host cells, and use a functionally similar internal ribosome entry site (IRES) for translation. In addition, both viruses use NS5B RNA-dependent RNA polymerase and have a similar mechanism for virion maturation, assembly, and release . In one study, the J6/JFH strain (a chimeric HCV of genotype 2a) did not replicate in B or T lymphocytes, monocytes, macrophages, or dendritic cells, while it did replicate and produce infectious virus in Huh-7.5 cells ; whereas, another study found that Mo-DC infected with the JFH1 strain of HCV with an MOI of 1did not support HCV RNA replication or antigen production . In contrast to the present study, the mechanism responsible for the inability to produce infectious HCV was different, as BVDV viral RNA was replicated and translated into viral protein. This variation in HCV and BVDV replication may be due to their replication requirements, as HCV tropism is restricted to hepatocytes of humans and chimpanzees , while BVDV can replicate in epithelial and non-epithelial cells .
A trial utilizing influenza virus in mouse bone marrow-derived DC resulted in abortive replication. However, viral genome transcription and replication occurred for each gene segment along with the translation of viral hemagglutinin and nucleoprotein. Further the study using electron microscopy examination revealed that the resulting failure was associated with defective viral release, possibly due to insufficient synthesis or stability of one or more viral proteins required for release of the virus . Similarly, the current study showed the abortive replication of BVDV in Mo-DC. The exact mechanism of abortive BVDV replication in Mo-DC is not known. However future ultrastructural examination of BVDV infected Mo-DC using electron microscopy could be intriguing to understand the stage at which production of infectious virus is blocked (i.e. assembly, egress etc).
The current study showed that Mo-DC precursor monocytes produced infectious BVDV while Mo-DC failed to do so. It would be interesting to know the dynamics of Mo-DC differentiation/maturation and viral production. Previous studies have demonstrated that Mo-DC differentiated with GMCSF and IL-4 were immature and could be matured by proinflammatory cytokines, CD40L, IFN-γ or LPS [13, 25]. There has been little work done on effect of BVDV strains on Mo-DC maturation. While it is well established that cp BVDV induced more IFN-γ than ncp BVDV in vitro [26, 27], it is likely that cp BVDV strains induce Mo-DC maturation more than ncp BVDV strains in vitro. To confirm this hypothesis, a future study would be helpful using different BVDV strains along with lipopolysaccharide (LPS) as maturation differentiation control.
One of the factors that may affect the production of virus in Mo-DC is interaction with other cell populations. In this in vitro study, isolated cell populations were used; whereas, testing in the in vivo environment may result in different outcomes. Interaction of Mo-DC with T cells in vivo may trigger the production of infectious virus. In one study, the interaction of antigen presenting cells (APC) with T cells enhanced HCV infection, as the interaction of APC with T cells led to activation of T cells . Activated T cells produce interleukin-2 (IL-2), a substance that enhances CD81 expression in thymocytes . Expression of CD81 on thymocytes facilitates the entry of HCV into cells, and thus, its replication . Similarly, co-stimulation through interaction of CD28 and B7 increased HIV type 1 replication . Therefore, it stands to reason that other cell populations could be affected. For example, the macrophage may become infected by phagocytizing the apoptotic Mo-DC containing viral RNA and may then begin producing infectious BVDV. As these probabilities are theoretical, exploration using T cell activation assay or in vivo trials would be beneficial.
The cell susceptibility can greatly be influenced with changes in its surface receptor expression, particularly receptors that mediate virus entry. BVDV infects cells via Receptor-mediated endocytosis using the LDL receptor . Given that oxidized LDL has been demonstrated to promote differentiation of dendritic cells from monocytes in other species , it would be interesting to explore the dynamics of LDL receptor expression during monocyte to Mo-DC differentiation with respect to cell infectivity and virus production. These future studies could also explore the effect of different BVDV on LDL expression for better understanding of the BVDV pathogenesis.
In the current study, infection with BVDV affected cell surface marker expression on Mo-DC. The cp BVDV1b-TGAC strain enhanced MHCI, MHCII, and CD86 expression, while ncp strains of BVDV (i.e., ncp BVDV2a 1373, ncp BVDV2a 28508-5, and ncp BVDV1b TGAN) reduced the MHCI, MHCII, and CD86 expression during the course of infection. In another study that used bovine monocyte-derived macrophages (MDM), MHCI was up-regulated following infection with cp BVDV and was down-regulated in cells infected with a ncp strain of BVDV, while both cp and ncp strains down-regulated the MHCII expression in MDM . The difference in MHCII results between that study and the present study may be due to a difference in the cell types chosen for analysis, as the Mo-DC reported here were non-adherent CD14- cells, while the MDM were adherent CD14+ cells. A study using differential detergent fractionation (DDF) analysis of bovine monocytes showed that 53 bovine proteins involved in the immune function of professional APC were altered following BVDV infection. These altered molecules include adhesion 151 molecules, toll-like receptors (TLR 1, 6, and 8), antigen uptake, MHC I and II, cytokines, and growth factor synthesis molecules . The above study supported the current finding that showed the alteration of MHCI, MHCII and CD86 expression in BVDV infected Mo-DC. The above study also showed the alteration of cell adhesion molecules and capability of antigen uptake by APC, that may affect the Mo-DC infectivity and ultimately virus producing capability that need to be explored.
The changes noted in surface marker expression on Mo-DC could be due to differences in interferon expression between cp and ncp biotypes of BVDV. Type 1 IFN plays an important role in up-regulation of MHCI expression , thus, reduced type 1 IFN production in Mo-DC infected with ncp BVDV may explain the down-regulation of MHCI and MHCII. Similarly, up-regulation of MHCI and MHCII in Mo-DC infected with cp BVDV may be due to an increase in type 1 IFN [26, 35]. It would be interesting to validate the effect of Type 1 IFN on cell surface marker expression by using poly I:C treated cells as control. The previous study showed that ncp BVDV inhibited of IFN induction was a result of blocking the IRF-3 pathway . It may be possible that BVDV affects other pathways such as the melanoma differentiation-associated gene 5 (MDA5) signaling pathway, which will need to be explored for better understanding of BVDV pathogenesis. Alternatively, infection may have a direct effect on MHC folding and assembly, and consequently, expression. For example, infection with HCV changed MHCI expression in infected cultured cancerous cells lines (i.e., human hepatoma and mouse lymphoma cells) via disruption in MHCI protein folding and assembly [36–38]. This study found that HCV replicons induced endoplasmic reticulum (ER) stress that resulted from a decline in protein glycosylation. The decrease in protein glycosylation disrupted protein folding and prevented the assembly of MHCI molecules. Reduced assembly of MHCI molecules ultimately resulted in MHCI down-regulation. Similarly, Human Immunodeficiency Virus (HIV) was shown to reduce MHCI and CD86 expression via the Nef protein in a human embryonic kidney cell line (293 T), the human monocytic U937 cell line, as well as in mouse macrophages and dendritic cells. The Nef protein binds and endocytoses MHCI molecules by the ARF6 pathway, thus resulting in reduced MHCI expression [39, 40]. The mechanism by which BVDV infection alters cell surface marker expression needs to be explored, as this information would provide a better understanding of immunosuppression caused by BVDV.