Participation of shrimp pva-miR-166 in hemocyte homeostasis by modulating apoptosis-related gene PvProsaposin during white spot syndrome virus infection.

Tiny controllers referred to as microRNAs (miRNAs) impede the expression of genes to modulate biological processes. In invertebrates, particularly in shrimp as a model organism, it has been demonstrated that miRNAs play a crucial role in modulating innate immune responses against viral infection. By analyzing small RNAs, we identified 60 differentially expressed miRNAs (DEMs) in Penaues vannamei hemocytes following infection with white spot syndrome virus (WSSV). We predicted the target genes of WSSV-responsive miRNAs, shedding light on their participation in diverse biological pathways. We are particularly intrigued by pva-miR-166, which is the most notably elevated miRNA among 60 DEMs. At 24 h post-infection (hpi), the negative correlation between the expression of pva-miR-166 and its target gene, PvProsaposin, was evident and their interaction was confirmed by a reduction in luciferase activity in vitro. Suppression of PvProsaposin in unchallenged shrimp led to decreased survival rates, reduced total hemocyte count (THC), and increased caspase 3/7 activity, suggesting its significant role in maintaining hemocyte homeostasis. In WSSV-infected shrimp, a lower number of hemocytes corresponded to a lower WSSV load, but higher shrimp mortality was observed when PvProsaposin was suppressed. Conformingly, the introduction of the pva-miR-166 mimic to WSSV-infected shrimp resulted in decreased levels of PvProsaposin transcripts, a significant loss of THC, and an increase in the hemocyte apoptosis. Taken together, we propose that pva-miR-166 modulates hemocyte homeostasis during WSSV infection by suppressing the PvProsaposin, an anti-apoptotic gene. PvProsaposin inhibition disrupts hemocyte homeostasis, rendering the shrimp's inability to withstand WSSV invasion.IMPORTANCEGene regulation by microRNAs (miRNAs) has been reported during viral infection. Furthermore, hemocytes serve a dual role, not only producing various immune-related molecules to combat viral infections but also acting as a viral replication site. Maintaining hemocyte homeostasis is pivotal for the shrimp's survival during infection. The upregulated miRNA pva-miR-166 could repress PvProsaposin expression in shrimp hemocytes infected with WSSV. The significance of PvProsaposin in maintaining hemocyte homeostasis via apoptosis led to reduced survival rate, decreased total hemocyte numbers, and elevated caspase 3/7 activity in PvProsaposin-silenced shrimp. Additionally, the inhibitory ability of pva-miR-166-mimic and dsRNA-PvProsaposin on the expression of PvProsaposin also lowered the THC, increases the hemocyte apoptosis, resulting in a lower WSSV copy number. Ultimately, the dysregulation of the anti-apoptotic gene PvProsaposin by pva-miR-166 during WSSV infection disrupts hemocyte homeostasis, leading to an immunocompromised state in shrimp, rendering them incapable of surviving WSSV invasion.

PmKuSPI is regulated by pmo-miR-bantam and contributes to hemocyte homeostasis and viral propagation in shrimp.

The Kunitz-type serine protease inhibitor (KuSPI) is a low molecular weight protein that plays a role in modulating a range of biological processes. In Penaeus monodon, the PmKuSPI gene has been found to be highly expressed in the white spot syndrome virus (WSSV)-infected shrimp and is predicted to be regulated by a conserved microRNA, pmo-miR-bantam. We reported that, despite being upregulated at the transcriptional level, the PmKuSPI protein was also upregulated after WSSV infection. Silencing the PmKuSPI gene in healthy shrimp had no effect on phenoloxidase activity or apoptosis but resulted in a delay in the mortality of WSSV-infected shrimp as well as a reduction in the total hemocyte number and WSSV copies. According to an in vitro luciferase reporter assay, the pmo-miR-bantam bound to the 3'UTR of the PmKuSPI gene as predicted. In accordance with the loss of function studies using dsRNA-mediated RNA interference, the administration of the pmo-miR-bantam mimic into WSSV-infected shrimp lowered the expression of the PmKuSPI transcript and the PmKuSPI protein, as well as the WSSV copy number. According to these results, the protease inhibitor PmKuSPI is posttranscriptionally controlled by pmo-miR-bantam and plays a role in hemocyte homeostasis, which in turn affects shrimp susceptibility to WSSV infection.

White Spot Syndrome Virus-Induced Shrimp miR-315 Attenuates Prophenoloxidase Activation via PPAE3 Gene Suppression.

MicroRNAs (miRNAs), the small non-coding RNAs, play a pivotal role in post-transcriptional gene regulation in various cellular processes. However, the miRNA function in shrimp antiviral response is not clearly understood. This research aims to uncover the function of pmo-miR-315, a white spot syndrome virus (WSSV)-responsive miRNAs identified from Penaeus monodon hemocytes during WSSV infection. The expression of the predicted pmo-miR-315 target mRNA, a novel PmPPAE gene called PmPPAE3, was negatively correlated with that of the pmo-miR-315. Furthermore, the luciferase assay indicated that the pmo-miR-315 directly interacted with the target site in PmPPAE3 suggesting the regulatory role of pmo-miR-315 on PmPPAE3 gene expression. Introducing the pmo-miR-315 into the WSSV-infected shrimp caused the reduction of the PmPPAE3 transcript level and, hence, the PO activity activated by the PmPPAE3 whereas the WSSV copy number in the shrimp hemocytes was increased. Taken together, our findings state a crucial role of pmo-miR-315 in attenuating proPO activation via PPAE3 gene suppression and facilitating the WSSV propagation in shrimp WSSV infection.