Type II Grass Carp Reovirus Infects Leukocytes but Not Erythrocytes and Thrombocytes in Grass Carp (Ctenopharyngodon idella)

Abstract

Grass carp reovirus (GCRV) causes serious losses to the grass carp industry. At present, infectious tissues of GCRV have been studied, but target cells remain unclear. In this study, peripheral blood cells were isolated, cultured, and infected with GCRV. Using quantitative real-time polymerase chain reaction (qRT-PCR), Western Blot, indirect immunofluorescence, flow cytometry, and transmission electron microscopy observation, a model of GCRV infected blood cells in vitro was established. The experimental results showed GCRV could be detectable in leukocytes only, while erythrocytes and thrombocytes could not. The virus particles in leukocytes are wrapped by empty membrane vesicles that resemble phagocytic vesicles. The empty membrane vesicles of leukocytes are different from virus inclusion bodies in C. idella kidney (CIK) cells. Meanwhile, the expression levels of IFN1, IL-1β, Mx2, TNFα were significantly up-regulated in leukocytes, indicating that GCRV could cause the production of the related immune responses. Therefore, GCRV can infect leukocytes in vitro, but not infect erythrocytes and thrombocytes. Leukocytes are target cells in blood cells of GCRV infections. This study lays a theoretical foundation for the study of the GCRV infection mechanism and anti-GCRV immunity.

Keywords: C. idella kidney (CIK) cells; erythrocytes; grass carp (Ctenopharyngodon idella); grass carp reovirus (GCRV); leukocytes; thrombocytes; virus inclusion bodies (VIBs).

Figure 1

Isolation of leukocytes in grass carp. (A) Grass carp peripheral blood cells were subjected to stratification and microscopy examination (scale bar: 200 μm). E: erythrocytes; T: thrombocytes; L: lymphocytes; N: neutrophils. (B) Analysis of leukocyte groups by flow cytometry. Group P1 denotes lymphocytes, accounting for about 83%. (C) Morphology of lymphocytes with Giemsa staining by light microscopy (scale bar: 100 μm). LL: large lymphocytes; SL: small lymphocytes. (D) Analysis of thrombocyte groups by flow cytometry. Group P2 is thrombocytes, accounting for about 57%. (E) Morphology of thrombocytes with Giemsa staining by light microscopy (scale bar: 100 μm).

Figure 2

After grass carp reovirus II (GCRV-II) infection, leukocytes, thrombocytes, erythrocytes, and C. idella kidney (CIK) cells were detected. (A) PCR results showed that leukocytes and CIK cells detected GCRV-positive with VP4 primer and EF1α as an internal reference, but erythrocytes and thrombocytes detected GCRV-negative. (B) Western blot results showed that with VP4 polyclonal antibody as primary antibody and β-actin as an internal reference, leukocytes and CIK cells detected GCRV-positive after infection, but erythrocytes and thrombocytes detected GCRV-negative. (C,D) Quantitative primers of VP4 and VP56 were used for qRT-PCR detection, respectively. The viral load of GCRV at 12 h, 24 h, 36 h, and 48 h after GCRV infection was detected, the control group was 0h after GCRV infection. Data of reporter assays and qPCR are shown as mean ± standard deviation (SD) of 6 wells of cell per group and are from one experiment representative of three independent experiments. Significance was calculated in relation to the control group. * p < 0.05, ** p < 0.01 (two tailed Student’s t-tests). The relative transcription levels were normalized to the transcription level of EF1α gene and are represented as fold induction relative to the transcription level in control cells, which was set to 1.

Figure 3

Leukocytic antiviral responses to GCRV-II infection. Expression of genes involved in antiviral responses was measured by qRT-PCR, use EF1α as an internal reference gene. The expression levels in infected leukocytes in 6-well plates were challenged with GCRV for 0 h, 12 h, 24 h, 36 h, and 48 h. The relative increase for IFN1 (A), Mx2 (B), IL-1β (C), and TNFα (D) is shown. (E) The expression levels of IFN1 in infected erythrocytes in 6-well plates were challenged with GCRV for 0 h, 12 h, 24 h, 36 h, and 48 h. (F) The expression levels of IFN1 in infected thrombocytes in 6-well plates were challenged with GCRV for 0 h, 12 h, 24 h, 36 h, and 48 h. Data of reporter assays and qPCR are shown as mean ± SD of 6 wells of cell per group and are from one experiment representative of three independent experiments. Significance was calculated in relation to the control group. * p < 0.05 (two tailed Student’s t-tests). The relative transcription levels were normalized to the transcription level of EF1α gene and are represented as fold induction relative to the transcription level in control cells, which was set to 1.

Figure 4

Several different morphogenesis of GCRV-II in CIK cells. (A) Mature virions arranged neatly like a lattice, with a diameter of about 70–80 nm; (B) Scattered virions, released in the cytoplasm, with a diameter of about 70–80 nm; (C) Early virus inclusion bodies are relatively dense and contain a small number of virions, which are in the process of forming; (D) Late VIBs, which are thick and have a large number of virions, are in the process of generating.

Figure 5

Transmission electron microscope observation of leukocytes, erythrocytes, and thrombocytes after infection with GCRV-II virus. (A,B) are the virus inclusion bodies (VIBs) in CIK cells infected with the virus. The structure is dense and contains a large number of virions in the process of being formed, without apparent membrane structure outside; (C,D) are virions in infected leukocytes that exist in the cytoplasm in the form of a relatively obvious envelope structure, which envelops a large number of virions, and the interior is not dense, showing a vacuole structure; (E) and (F) are respectively infected erythrocytes and thrombocytes, both cells have no noticeable pathological changes, and their constructions are complete.

Figure 6

Immunofluorescence of GCRV-II infected peripheral blood cells. (A) Fluorescent labeling of the GCRV-II vp4-protein (green) in ex vivo infected leukocytes, the nuclei were stained with Hoechst (blue). Confocal microscopy of different staining patterns in the cytoplasm, including a few inclusions and scattered granular staining in the perinuclear region (scale bar: 10 μm). (B) Confocal microscopy images showing viral inclusions in the perinuclear area. (C) Fluorescent labeling of the GCRV-II vp4-protein (green) in ex vivo infected erythrocytes, the nuclei were stained with Hoechst (blue). Confocal microscopy images showing erythrocytes were GCRV negative (scale bar: 10 μm). (D) Fluorescent labeling of the GCRV-II vp4-protein (green) in infected CIK cells, the nuclei were stained with Hoechst (blue). Confocal microscopy images showing many virus inclusions were found in the cytoplasm (scale bar: 20 μm).

Figure 7

GCRV-positive leukocytes, GCRV-negative erythrocytes and GCRV-positive CIK cells detected by flow cytometry. Flow cytometry result from the intracellular staining for GCRV vp4 protein from GCRV infected cell (green) and control cell (blue) at 0 h, 12 h, 24 h, 36 h and 48 h post infection. The fluorescence intensity (GCRV vp4) is shown on the X-axis and cell count on the y-axis counting 10,000 cells per sample. (A) In ex vivo infected leukocytes, GCRV-positive rates were 0.32%, 23.47%, 33.02%, 45.95%, 64.60% at 0 h, 12 h, 24 h, 36 h and 48 h, respectively. (B) In ex vivo infected erythrocytes, GCRV-positive rates were 0.01%, 0.64%, 0.38%, 0.45%, 1.04% at 0h, 12h, 24h, 36h and 48h, respectively. (C) In infected CIK cells, GCRV-positive rates were 0.24%, 38.85%, 43.96%, 64.35%, 86.78% at 0 h, 12 h, 24 h, 36 h and 48 h, respectively.