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. 2022 Jun 5;14(6):1227.
doi: 10.3390/v14061227.

Antiviral Activities of Green Tea Components against Grouper Iridovirus Infection In Vitro and In Vivo

Pengfei Li  1   2 , Shuaishuai Huang  1 , Shuangyan Xiao  2 , Youhou Xu  1 , Xinxian Wei  3 , Jun Xiao  3 , Zhongbao Guo  3 , Qing Yu  2 , Mingzhu Liu  2
Affiliations

Antiviral Activities of Green Tea Components against Grouper Iridovirus Infection In Vitro and In Vivo

Pengfei Li et al. Viruses. .

Abstract

(1) Background: Singapore grouper iridovirus (SGIV) can cause extensive fish deaths. Therefore, developing treatments to combat virulent SGIV is of great economic importance to address this challenge to the grouper aquaculture industry. Green tea is an important medicinal and edible plant throughout the world. In this study, we evaluated the use of green tea components against SGIV infection. (2) Methods: The safe working concentrations of green tea components were identified by cell viability detection and light microscopy. Additionally, the antiviral activity of each green tea component against SGIV infection was determined with light microscopy, an aptamer (Q5c)-based fluorescent molecular probe, and reverse transcription quantitative PCR. (3) Results: The safe working concentrations of green tea components were green tea aqueous extract (GTAE) ≤ 100 μg/mL, green tea polyphenols (TP) ≤ 10 μg/mL, epigallocatechin-3-gallate (EGCG) ≤ 12 μg/mL, (-)-epigallocatechin (EGC) ≤ 10 μg/mL, (-)-epicatechin gallate (EGC) ≤ 5 μg/mL, and (-)-epicatechin (EC) ≤ 50 μg/mL. The relative antiviral activities of the green tea components determined in terms of MCP gene expression were TP > EGCG > GTAE > ECG > EGC > EC, with inhibition rates of 99.34%, 98.31%, 98.23%, 88.62%, 73.80%, and 44.31%, respectively. The antiviral effect of aptamer-Q5c was consistent with the results of qPCR. Also, TP had an excellent antiviral effect in vitro, wherein the mortality of fish in only the SGIV-injection group and TP + SGIV-injection group were 100% and 11.67%, respectively. (4) Conclusions: In conclusion, our results suggest that green tea components have effective antiviral properties against SGIV and may be candidate agents for the effective treatment and control of SGIV infections in grouper aquaculture.

Keywords: EGCG; antiviral activity; green tea component; grouper iridovirus; tea polyphenol.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Green tea and structures of major components (EGCG, ECG, EGC, and EG).
Figure 2
Figure 2
Determining the working concentrations of green tea components. (A) Grouper spleen (GS) cells were incubated with different concentrations of green tea components for 48 h and then observed with light microscopy. Morphological changes were observed in the GS cells incubated with green tea aqueous extract (GTAE), TP (tea polyphenols), epigallocatechin-3-gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), and epicatechin (EC). (B) Cell survival analysis with CCK-8 solution: GS cells were incubated with different concentrations of green tea components. The results of comparisons with p < 0. 05 were considered to represent statistically significant differences (* p < 0. 05, ** p < 0. 01, NS is no significant difference.). The same below.
Figure 3
Figure 3
RT qPCR was used to analyze the antiviral effects of green tea components. ** indicates p < 0.01.
Figure 4
Figure 4
The antiviral activities of green tea components against SGIV observed with light microscopy at 48 h. GS cells incubated with SGIV and diluted green tea components (GTAE at 100, 50, or 25 μg/mL; TP at 10, 5, or 2.5 μg/mL; and EGCG at 12, 6, or 3 μg/mL) constituted the test groups. GS cells infected with SGIV-only constituted the positive control group. GS cells incubated with L15 medium only constituted the negative control group. Each green tea component had the greatest antiviral effect at its safe working concentration (GTAE at 100 μg/mL, TP at 10 μg/mL, and EGCG at 12 μg/mL).
Figure 5
Figure 5
Antiviral activities of green tea components against SGIV tested with qPCR at 48 h. GS cells incubated with SGIV and diluted green tea components (GTAE at 100, 50, or 25 μg/mL; TP at 10, 5, or 2.5 μg/mL; and EGCG at 12, 6, or 3 μg/mL) constituted the test groups. Untreated SGIV-infected GS cells constituted the positive control group. The antiviral effects were analyzed as the inhibition of MCP and VP19 expression. ** indicates p < 0.01.
Figure 6
Figure 6
Antiviral activities of green tea components against SGIV evaluated with Q5c-AFMP at 48 h. GS cells incubated with SGIV and the diluted green tea components (GTAE at 100, 50, or 25 μg/mL; TP at 10, 5, or 2.5 μg/mL; and EGCG at 12, 6, or 3 μg/mL) constituted the test groups. GS cells infected with SGIV only constituted the positive control group. Antiviral effects were analyzed as the fluorescence intensity of cells. ** indicates p < 0.01.
Figure 7
Figure 7
Percentage inhibition analysis of each green tea component against SGIV infection. The percentage inhibition of SGIV infection by each green tea component was 99.44% (GTAE), 99.79% (TP), and 99.99% (EGCG) when measured as MCP expression and 98.60% (GTAE), 99.83% (TP), and 99.99% (EGCG) when measured as VP19 gene expression.
Figure 8
Figure 8
Antiviral activity of green tea component TP against SGIV in vivo. Hybrid groupers were injected intraperitoneally with SGIV mixed with TP at its safe working concentration of 10 μg/mL. Spleen tissues were analyzed for antiviral activity at 24, 48, and 72 hpi. ** indicates p < 0.01.
Figure 9
Figure 9
Cumulative mortality over 10 days.
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