Specific regulation of the chemokine response to viral hemorrhagic septicemia virus at the entry site

Abstract

The fin bases constitute the main portal of rhabdovirus entry into rainbow trout (Oncorhynchus mykiss), and replication in this first site strongly conditions the outcome of the infection. In this context, we studied the chemokine response elicited in this area in response to viral hemorrhagic septicemia virus (VHSV), a rhabdovirus. Among all the rainbow trout chemokine genes studied, only the transcription levels of CK10 and CK12 were significantly upregulated in response to VHSV. As the virus had previously been shown to elicit a much stronger chemokine response in internal organs, we compared the effect of VHSV on the gills, another mucosal site which does not constitute the main site of viral entry or rhabdoviral replication. In this case, a significantly stronger chemokine response was triggered, with CK1, CK3, CK9, and CK11 being upregulated in response to VHSV and CK10 and CK12 being down-modulated by the virus. We then conducted further experiments to understand how these different chemokine responses of mucosal tissues could correlate with their capacity to support VHSV replication. No viral replication was detected in the gills, while at the fin bases, only the skin and the muscle were actively supporting viral replication. Within the skin, viral replication took place in the dermis, while viral replication was blocked within epidermal cells at some point before protein translation. The different susceptibilities of the different skin layers to VHSV correlated with the effect that VHSV has on their capacity to secrete chemotactic factors. Altogether, these results suggest a VHSV interference mechanism on the early chemokine response at its active replication sites within mucosal tissues, a possible key process that may facilitate viral entry.

Fig. 1.

Levels of Mx gene transcription in response to VHSV bath infection in fin bases (A) and gills (B). Samples were collected from both infected and mock-infected controls after 1 or 3 days of VHSV infection; RNA was obtained, and the levels of transcription of the Mx gene were determined by real-time PCR. Individual data (circles) were analyzed in triplicate and are shown as the mean gene expression (black bars) relative to the expression of endogenous control EF-1α. *, relative mean expression significantly higher than the relative mean expression in the respective control (P < 0.05).

Fig. 2.

Levels of VHSV N gene transcription in the fin bases in response to VHSV. Fin bases were collected from both infected and mock-infected controls after 1 or 3 days of VHSV infection; RNA was obtained, and the levels of transcription of the N viral gene were determined by real-time PCR. Individual data (circles) were analyzed in triplicate and are shown as the mean gene expression (black bar) relative to the expression of endogenous control EF-1α. *, relative mean expression significantly higher than the relative mean expression in the respective control (P < 0.05).

Fig. 3.

Immunohistochemical staining of fin bases infected with VHSV. Although the immunohistochemical staining was performed with both control and infected fish obtained at days 1, 3, and 6 postinfection, no significant differences in the level or distribution of staining were observed in infected fish at different days postinfection. Therefore, samples taken at day 1 postinfection are shown.(a) Control fin base area stained with hematoxylin-eosin corresponding to the area sampled in chemokine expression studies (magnification, ×10). (b, d, and f) Images from control fish showing dermis, epidermis, and muscle, respectively, in which no staining was observed (magnification, ×20). (c, e, and g) Images from infected fish showing dermis, epidermis, and muscle, respectively (magnification, ×20). Specific staining for VHSV in red was always observed in muscle and dermis but not in epidermis.

Fig. 4.

Cultures were established either from complete skin in which both epidermal cells and dermal cells were present or exclusively from epidermal cells. These two types of skin cultures were infected in vitro with VHSV or mock infected to determine their susceptibility to VHSV. (A) Levels of transcription of the viral N gene and Mx gene transcription determined through real-time PCR in complete skin or epidermis cultures after 3 days of incubation with the virus in vitro. Data are shown as gene expression relative to the expression of endogenous control EF-1α obtained in individual cultures or as stimulation indexes in the case of Mx, obtained by dividing the levels of Mx expression observed in response to VHSV to those observed in the respective mock-infected culture. (B) Detection of N viral protein through Western blotting in fin explants or epidermis cell cultures infected with VHSV (V) or mock infected (C) in vitro at day 3 postinfection. (C) Viral titer obtained in four independent cultures after 7 days of infection with VHSV at a final concentration of 5 × 10⁴ TCID₅₀/ml.

Fig. 5.

Levels of transcription of the different chemokine genes in the different skin cultures established. RNA was extracted from either complete skin cultures or epidermis cell cultures from which supernatants were collected, and the levels of transcription of these chemokines were studied through real-time PCR. Data are shown as mean chemokine gene expression ± standard deviation relative to the expression of endogenous control EF-1α of four independent cultures. ND, not detected.

Fig. 6.

Effect of VHSV on the capacity of the different skin cultures to secret chemotactic factors. Cultures were established either from complete skin in which both epidermal cells and dermal cells were present or exclusively from epidermal cells. These two types of skin cultures were infected in vitro with VHSV or mock infected and incubated for 3 days at 14°C. At this point, supernatants (SN) from VHSV-infected cultures and from mock-infected cultures were collected to determine the capacity of these supernatants to induce migration of autologous PBLs by comparing the migration to the migration observed toward medium alone (Control) or medium with VHSV. The chemotaxis assay was performed as described in Materials and Methods, and fluorescence-activated cell sorting analysis was used to enumerate the number of migrating cells. Experiments were performed in triplicate, and data are shown as the mean number (± standard deviation) of migrating cells obtained in individual rainbow trout (RT). *, migration levels toward supernatants from infected cultures significantly different from migration levels obtained with supernatants from mock-infected cultures (P < 0.05).