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

(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.

Molecular characterization and functional analysis of scavenger receptor class B from black tiger shrimp (Penaeus monodon)

BACKGROUND: Scavenger receptor class B (SRB) is a multifunctional protein in animals that participates in physiological processes, including recognition of a wide range of ligands. Astaxanthin is a major carotenoid found in shrimp. However, the molecular mechanism of astaxanthin and SRB protein binding has not been reported. RESULTS: In the present study, a member of the SRB subfamily, named PmSRB, was identified from the transcriptome of black tiger shrimp (Penaeus monodon). The open reading frame of PmSRB was 1557 bp in length and encoded 518 amino acids. The structure of PmSRB included a putative transmembrane structure at the N-terminal region and a CD36 domain. Multiple sequence alignment indicated that the CD36 domain were conserved. Phylogenetic analysis showed four separate branches (SRA, SRB, SRC, and croquemort) in the phylogenetic tree and that PmSRB was clustered with SRB of Eriocheir sinensis. Quantitative real-time polymerase chain reaction showed that the PmSRB gene was widely expressed in all tissues tested, with the highest expression level observed in the lymphoid organ and brain. Subcellular localization analysis revealed that PmSRB-GFP (green fluorescent protein) fusion proteins were predominantly localized in the cell membrane. The recombinant proteins of PmSRB showed binding activities against astaxanthin in vitro. CONCLUSIONS: PmSRB was identified and characterized in this study. It is firstly reported that PmSRB may take as an important mediator of astaxanthin uptake in shrimp.

Streptomyces qinzhouensis sp. nov., a mangrove soil actinobacterium.

A novel Streptomyces strain (SSL-25T) was isolated from mangrove soil sampled at QinzhouBay, PR China. The isolate was observed to be Gram-stain-positive and to form greyish-white aerial mycelia that differentiated into straight spore chains with smooth-surfaced spores on International Streptomyces Project 2 medium. The cell-wall peptidoglycan was determined to contain ll-diaminopimelicacid. The cell-wall sugars were glucose and mannose. The predominant menaquinones were MK-9 (H6), MK-9 (H8) and MK-9 (H4). The major polar lipids contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and several unidentified phospholipids. The predominant cellular fatty acids were C16:0, iso-C16:0 and summed feature 3 (C16:1ω7c/C16:1ω6c). The genome size of strain SSL-25T was 8.1 Mbp with a G+C content of 71.5 mol%. Phylogenetic analysis indicated that strain SSL-25T is closely related to Streptomyces tsukubensis NRRL 18488T (99.4 % sequence similarity). However, the digital DNA-DNA hybridization (39.8 %) and average nucleotide identity (91.3 %) values between them showed that it represents a distinct species. Furthermore, the results of morphological, physiological and biochemical tests allowed further phenotypic differentiation of strain SSL-25T from S. tsukubensis NRRL 18488T. Therefore, based on these results, it is concluded that strain SSL-25T represents a novel Streptomyces species, for which the name Streptomyces qinzhouensis sp. nov. is proposed. The type strain is SSL-25T (=CICC 11054T=JCM33585T).

Identification and functional characterization of a c-type lysozyme from Fenneropenaeus penicillatus.

Lysozyme is an important defense molecule of the innate immune system and possess high antimicrobial activities. In this study, a full-length c-type lysozyme cDNA (Fplysc) was cloned and characterized from Fenneropenaeus penicillatus. The cDNA contains an open reading frame of 477 bp encoding 158 amino acids, with 53-94% identity with those of other crustaceans. The recombinant Fplysc had antibacterial activities against Gram-positive bacteria (Streptococcus agalactiae and Micrococcus luteus) and Gram-negative bacteria (Vibrio alginolyticus and Escherichia coli), and showed antiviral activity against WSSV and IHHNV. The qRT-PCR analysis showed that Fplysc expression levels were most abundant in hemocytes and less in eyestalk. The expression levels of Fplysc were significantly upregulated in gill, intestine and hemocytes when challenged with WSSV and V. alginolyticus. Fplysc-silencling suppressed Fplysc expression in cephalothoraxes and increased mortality caused by WSSV and V. alginolyticus, and exogenous rFplysc led to a significant decrease of shrimp mortality by injecting rFplysc into Fplysc silenced shrimp, suggesting Fplysc is the important molecule in shrimp antimicrobial and antiviral response. In conclusion, the results provide some insights into the function of Fplysc in shrimp against bacterial and viral infection.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) participates in peroxinectin gene expression in Fenneropenaeus penicillatus.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an important cytoplasm signal adaptor that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. In the study, the full-length cDNA of a TRAF6 homolog (FpTRAF6) was identified from Fenneropenaeus penicillatus. The full-length cDNA of FpTRAF6 is 2033 bp long, with an open reading frame (ORF) encoding a putative protein of 594 amino acids, including a RING type Zinc finger, two TRAF-type Zinc fingers, and a conserved C-terminal meprin and TRAF homology (MATH) domain. The overall amino acid sequence identity between FpTRAF6 and other TRAF6s ranged from 62.7 to 94.1% for crustaceans and from 45.6 to 59.3% for mollusca. Real-time qRT-PCR indicated that FpTRAF6 was constitutively expressed in various tissues of F. penicillatus. The temporal expression patterns of FpTRAF6 mRNA were different in the different tissues after microbial challenge. FpTRAF6 was downregulated in the heart, no obvious changes in the gill, intestine and hemocytes, and upregulated in other tested tissues after WSSV challenge. After V. alginolyticus injection, FpTRAF6 was downregulated in the heart and intestine, upregulated in the gill, lymphoid organ and hematopoietic organ, and no obvious changes in other tested tissues. RNAi assay was carried out to investigate the function of FpTRAF6. The results showed that silencing FpTRAF6 gene could inhibit peroxinectin expression in vivo, and enhance the sensitivity of shrimps to WSSV and V. alginolyticus challenge, suggesting FpTRAF6 could play a positive role against bacterial and viral pathogens. In conclusion, the results of the study provide some insights into the function of FpTRAF6 in activating TLRs signaling pathway and the host defense against invading pathogens.