WSSV early protein WSSV004 enhances viral replication by suppressing LDH activity.

White spot syndrome virus (WSSV) is known to upregulate glycolysis to supply biomolecules and energy for the virus's replication. At the viral genome replication stage, lactate dehydrogenase (LDH), a glycolytic enzyme, shows increased activity without any increase in expression. In the present study, yeast 2-hybrid screening was used to identify WSSV proteins that interacted with LvLDH isoform 1 and 2, and these included the WSSV early protein WSSV004. The interaction between WSSV004 and LvLDH1/2 was confirmed by co-immunoprecipitation. Immunofluorescence showed that WSSV004 co-localized with LvLDH1/2 in the cytoplasm. dsRNA silencing experiments showed that WSSV004 was crucial for WSSV replication. However, although WSSV004 silencing led to the suppression of total LvLDH gene expression during the viral late stage, there was nevertheless a significant increase in LvLDH activity at this time. We also used affinity purification-mass spectrometry to identify cellular proteins that interact with WSSV004, and found a total of 108 host proteins and 3 WSSV proteins with which it potentially interacts. Bioinformatics analysis revealed that WSSV004 and its interacting proteins might be responsible for various biological pathways during infection, including vesicular transport machinery and RNA-related functions. Collectively, our study suggests that WSSV004 serves as a multifunctional modulator to facilitate WSSV replication.

White spot syndrome virus (WSSV) modulates lipid metabolism in white shrimp.

In addition to the Warburg effect, which increases the availability of energy and biosynthetic building blocks in WSSV-infected shrimp, WSSV also induces both lipolysis at the viral genome replication stage (12 hpi) to provide material and energy for the virus replication, and lipogenesis at the viral late stage (24 hpi) to complete virus morphogenesis by supplying particular species of long-chain fatty acids (LCFAs). Here, we further show that WSSV causes a reduction in lipid droplets (LDs) in hemocytes at the viral genome replication stage, and an increase in LDs in the nuclei of WSSV-infected hemocytes at the viral late stage. In the hepatopancreas, lipolysis is triggered by WSSV infection, and this leads to fatty acids being released into the hemolymph. ß-oxidation inhibition experiment reveals that the fatty acids generated by WSSV-induced lipolysis can be diverted into ß-oxidation for energy production. At the viral late stage, WSSV infection leads to lipogenesis in both the stomach and hepatopancreas, suggesting that fatty acids are in high demand at this stage for virion morphogenesis. Our results demonstrate that WSSV modulates lipid metabolism specifically at different stages to facilitate its replication.

White Spot Syndrome Virus Triggers a Glycolytic Pathway in Shrimp Immune Cells (Hemocytes) to Benefit Its Replication.

White spot syndrome virus (WSSV) is the causative agent of a shrimp disease that inflicts in huge economic losses in shrimp-farming industry. WSSV triggers aerobic glycolysis in shrimp immune cells (hemocytes), but how this virus regulates glycolytic enzymes or pathway is yet to be characterized. Therefore, mRNA levels and activity of four important glycolytic enzymes, Hexokinase (HK), Phosphofructokinase (PFK), Pyruvate kinase (PK), and Lactate dehydrogenase (LDH), were measured in WSSV-infected shrimp hemocytes. Gene expression of HK and PFK, but not LDH or PK, was increased at the viral genome replication stage (12 hpi); furthermore, activity of these enzymes, except HK, was concurrently increased. However, there was no increased enzyme activity at the viral late stage (24 hpi). In vivo dsRNA silencing and glycolysis disruption by 2-DG further confirmed the role of glycolysis in virus replication. Based on tracing studies using stable isotope labeled glucose, glycolysis was activated at the viral genome replication stage, but not at the viral late stage. This study demonstrated that WSSV enhanced glycolysis by activating glycolytic enzyme at the viral genome replication stage, providing energy and biomolecules for virus replication.

Penaeus vannamei serine proteinase inhibitor 7 (LvSerpin7) acts as an immune brake by regulating the proPO system in AHPND-affected shrimp.

Acute hepatopancreatic necrosis disease (AHPND) is a recently emerged disease in aqua cultured shrimp that is caused by virulent strains of Vibrio parahaemolyticus (VP). Our previous study used transcriptomics to identify key pathogenic factors in the stomach of AHPND-infected shrimp (Litopenaeus vannamei), and here we used a different subset of the same data to construct a gene-to-gene expression correlation network to identify immune-responsive genes. LvSerpin7 was found to have the highest number of correlations after infection, and it also showed a significant increase in mRNA expression. LvSerpin7 is expressed in all tissues but its expression levels are highest in hemocytes. After successfully silencing LvSerpin7 transcript prior to AHPND challenge, mortality was significantly increased relative to the controls and reached 100% within 36 h post infection. Compared to the controls, the phenoloxidase (PO) activity also increased in both hemolymph and stomach. Recombinant LvSerpin7 inhibited shrimp PO activity in vitro, and we also found that rLvSerpin7 inhibited the growth of AHPND-causing bacteria. These results suggest that LvSerpin7 might reduce the toxic effects that result from unregulated activation of the PO defense system by AHPND-causing bacteria.