Small Molecule Inhibitors of White Spot Syndrome Virus: Promise in Shrimp Seedling Culture

Abstract

Rapid production of prawn (Litopenaeus vannamei) under artificial pressure can result in a series of obvious challenges and is vulnerable to serious losses related to aquatic environmental issues and the unrestrained outbreak of white spot syndrome (WSS). However, to date, there are no therapeutic strategies to contain the spread of the virus. Here, we synthesized 27 coumarin derivatives and evaluated their anti-white spot syndrome virus (WSSV) activity in L. vannamei larvae. We demonstrated that electron-withdrawing and electron-giving substituent groups play an important role in reducing toxicity and WSSV replication, respectively. Two coumarin C2 (2-amino-5-oxo-4-(p-tolyl)-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile) and C7 (2-amino-4-(4-chlorophenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile) were regarded as the most promising anti-WSSV compounds with maximum antiviral response <5% and median effective concentration <10 mg/L. The mortality of WSSV-infected larvae decreased by more than 60% after exposure to C2 and C7. With continuous immersion of C2 and C7 exchange, the mortality further decreased to 40% at 120 h. Additionally, C2 and C7 are the relatively stable in aquacultural water, making these agents suitable for use in inhibiting WSSV horizontal transmission in static aquaculture systems. These results showed the marked advantages of using C2 and C7 in the shrimp industry, and suggest that they hold potential for the treatment and prevention of WSSV infection in shrimp seedling culture.

Keywords: Litopenaeus vannamei; continuous immersion; coumarin; horizontal transmission; white spot syndrome virus.

Figure 1

The percent inhibition of the 27 coumarin derivatives on white spot syndrome virus (WSSV) replication in shrimp larvae is shown. (A) Schematic diagram describing the workflow. (B–D) Six-point dose-response curves for antiviral activity of coumarins are examined. The percent inhibition on quantification on copy number of viral genome is analyzed by RT-qPCR. Each value is represented as the mean ± SD normalized to values for no treatment.

Figure 1

The percent inhibition of the 27 coumarin derivatives on white spot syndrome virus (WSSV) replication in shrimp larvae is shown. (A) Schematic diagram describing the workflow. (B–D) Six-point dose-response curves for antiviral activity of coumarins are examined. The percent inhibition on quantification on copy number of viral genome is analyzed by RT-qPCR. Each value is represented as the mean ± SD normalized to values for no treatment.

Figure 2

C2 and C7 inhibited WSSV infection in shrimp larvae. (A) Schematic diagram describing the workflow. (B) The survival rate of WSSV-infected shrimp was analyzed in 0.31 to 10 mg/L treatments at 72 hpi. (C) Survivorship curve of WSSV-infected shrimp was analyzed in C2 and C7 up to 10 mg/L treatment. (D) Quantification on copy number of viral genome was tested by exposure to C2 and C7 up to 10 mg/L treatment at 24, 48, and 72 hpi, respectively. The virus-infected shrimps were immersed with the compounds and DMSO at the dose 0.2‰ (v/v). Each value is represented as the mean ± SD normalized to values for no treatment. The p value for each study was determined by Student’s t tests. ** p < 0.01.

Figure 3

Effect of C2 and C7 pre-exposure was evaluated on WSSV infection. (A) Schematic diagram describing the workflow. (B) Quantification on copy number of viral genome in shrimp larvae was tested in C2 and C7 pre-exposure to larvae for 1, 4, and 8 h, followed by WSSV infection. (C,D) Survivorship curve of WSSV-infected shrimp was analyzed in C2 and C7 up to 10 mg/L pre-exposure treatment. Each value is represented as the mean ± SD normalized to values for no treatment. The p value for each study was determined by Student’s t tests.

Figure 4

Antiviral activity of C2 and C7 was evaluated on preincubation with WSSV. (A) Schematic diagram describing the workflow. (B) Quantification on copy number of viral genome in shrimp larvae was tested in C2 and C7 preincubation with WSSV for 1, 2, and 4 h, followed by infection to larvae. (C,D) Survivorship curve of WSSV-infected shrimp was analyzed in C2 and C7 at the concentration of 10 mg/L in 1, 2, and 4 h preincubation. Each value is represented as the mean ± SD normalized to values for no treatment. The p value for each study was determined by Student’s t tests. ** p < 0.01; * p < 0.05.

Figure 5

Analysis on the related stability of C2 and C7 in aquacultural water at 28 °C. (A) Quantification on copy number of viral genome in shrimp larvae was tested in C2 and C7 for different water samples by RT-qPCR. Each value is represented as the mean ± SD normalized to values for no treatment. (B–E) Survivorship curve of WSSV-infected shrimp was analyzed in C2 and C7 at the concentration of 10 mg/L in 1, 2, 3, and 4 d water samples.

Figure 6

Continuous immersion of C2 and C7 exchange provides a strong protection on shrimp larvae against WSSV infection. (A) Schematic diagram describing the workflow. (B–F) Aquacultural water or fresh C2 and C7 medium were exchanged at 0, 1, 2, 3, and 4 days, respectively. Survivorship curves of WSSV-infected shrimp was analyzed. (G) The heat map indicated that quantification on copy number of viral genome was tested in every 8 h. Each value is represented as the mean ± SD normalized to values for no treatment.

Figure 7

Inhibition of C2 and C7 on WSSV horizontal transmission. Each value is represented as the mean ± SD normalized to values for no treatment. The p value for each study was determined by Student’s t tests. ** p < 0.01; * p < 0.05.

Figure 8

Synthetic route of coumarin derivatives A1–9, B1–9, and C1–9. Reagents and conditions: (a) 0.1 M SLS, H₂O, 60–65 °C, 5 h.