Synthesis and evaluation of polyamine carbon quantum dots (CQDs) in Litopenaeus vannamei as a therapeutic agent against WSSV

Abstract

White spot syndrome virus (WSSV) is the causative agent of white spot syndrome (WSS), a disease that has led to severe mortality rates in cultured shrimp all over the world. The WSSV is a large, ellipsoid, enveloped double-stranded DNA virus with a wide host range among crustaceans. Currently, the main antiviral method is to block the receptor of the host cell membrane using recombinant viral proteins or virus antiserum. In addition to interference with the ligand-receptor binding, disrupting the structure of the virus envelope may also be a means to combat the viral infection. Carbon quantum dots (CQDs) are carbonaceous nanoparticles that have many advantageous characteristics, including small size, low cytotoxicity, cheap, and ease of production and modification. Polyamine-modified CQDs (polyamine CQDs) with strong antibacterial ability have been identified, previously. In this study, polyamine CQDs are shown to attach to the WSSV envelope and inhibit the virus infection, with a dose-dependent effect. The results also show that polyamine CQDs can upregulate several immune genes in shrimp and reduce the mortality upon WSSV infection. This is first study to identify that polyamine CQDs could against the virus. These results, indeed, provide a direction to develop effective antiviral strategies or therapeutic methods using polyamine CQDs in aquaculture.

Figure 1

Identification of polyamine CQD anti-viral ability in vitro, by real-time PCR. The virus was treated with 20 or 1 ppm polyamine CQDs, and infected haemocytes. The expression of ie1 was used as a WSSV infection indicator and EF1-α was set as control. The experiment was repeated three times. Each bar represents the mean ± SD. The statistical significance was calculated using Student’s t-test. Significant differences (p < 0.05) in gene expression level between the compared groups are indicated with two asterisks. Triple asterisks indicated greater significant difference (p < 0.0005).

Figure 2

Cellular localizations of polyamine CQDs in WSSV by cryo-EM. (a) The virus treated with PBS (0 ppm, control), 1 or 20 ppm polyamine CQD. Red circles are polyamine CQDs. Scale bar: 100 nm. (b) Comparison of the number of polyamine CQDs around control and polyamine CQDs-containing virus. The statistical significance was calculated using Student’s t-test. Significant differences (p < 0.05) between the compared groups are indicated with asterisk. Double asterisks indicate very significant differences (p < 0.005).

Figure 3

The gene expressions of proPO in shrimp haemocyte after feeding polyamine CQD containing-feed, evaluated by real-time PCR. The experiment was repeated three times. Each bar represents the mean ± SD. The statistical significance was calculated using Student’s t-test. Very significant differences (p < 0.005) of gene expression level between the compared groups are indicated with double asterisks. Triple asterisks indicated greater significant difference (p < 0.0005).

Figure 4

The relative gene expressions of haemolymph clotting mechanism in shrimp haemocyte after feeding polyamine CQD containing-feed, evaluated by real-time PCR. (a) TGase and (b) CP gene expressions in shrimp haemocyte. The experiment was repeated three times. Each bar represents the mean ± SD. The statistical significance was calculated using Student’s t-test. Significant differences (p < 0.05) of gene expression level between the compared groups are indicated with double asterisks. The double asterisks indicated greater significant difference (p < 0.005).

Figure 5

The relative gene expressions of antimicrobial peptide system in shrimp haemocyte after feeding polyamine CQD containing-feed, evaluated by real-time PCR. (a) ALF, (b) Crustin, (c) Lyz, (d) PEN2, (e) PEN3 and (f) PEN4 gene expressions in shrimp haemocyte. The experiment was repeated three times. Each bar represents the mean ± SD. Significant differences (p < 0.05) of gene expression level between the compared groups are indicated with one asterisk. Double asterisks indicate very significant difference (p < 0.005).

Figure 6

The relative gene expressions of antioxidant defence mechanism in shrimp haemocyte after feeding polyamine CQD containing-feed, evaluated by real-time PCR. (a) SOD and (b) GPx gene expressions in shrimp haemocyte. The experiment was repeated three times. Each bar represents the mean ± SD. Significant differences (p < 0.05) of gene expression level between the compared groups are indicated with one asterisk.

Figure 7

The gene expressions of LvToll in shrimp haemocyte after feeding polyamine CQD contained-feed, evaluated by real-time PCR. The experiment was repeated three times. Each bar represents the mean ± SD. Significant differences (p < 0.05) of gene expression level between the compared groups are indicated with one asterisk.

Figure 8

Identification of CQD anti-viral ability in vivo. The experiment was done using 20 shrimps in each group and repeated 3 times. Each bar represents the mean ± SD.