The function of Toll pathway defense against bacterial infection has been well established in shrimp, however how this pathway responds to viral infection is still largely unknown. In this study, we report the Toll4-Dorsal-AMPs cascade restricts the white spot syndrome virus (WSSV) infection of shrimp. A total of nine Tolls from Litopenaeus vannamei namely Toll1-9 are identified, and RNAi screening in vivo reveals the Toll4 is important for shrimp to oppose WSSV infection. Knockdown of Toll4 results in elevated viral loads and renders shrimp more susceptible to WSSV. Furthermore, Toll4 could be a one of upstream pattern recognition receptor (PRR) to detect WSSV, and thereby leading to nuclear translocation and phosphorylation of Dorsal, the known NF-κB transcription factor of the canonical Toll pathway. More importantly, silencing of Toll4 and Dorsal contributes to impaired expression of a specific set of antimicrobial peptides (AMPs) such as anti-LPS-factor (ALF) and lysozyme (LYZ) family, which exert potent anti-WSSV activity. Two AMPs of ALF1 and LYZ1 as representatives are demonstrated to have the ability to interact with several WSSV structural proteins to inhibit viral infection. Taken together, we therefore identify that the Toll4-Dorsal pathway mediates strong resistance to WSSV infection by inducing some specific AMPs.

Emerging studies have indicated that some penaeidins restrict virus infection; however, the mechanism(s) involved are poorly understood. In the present study, we uncovered that penaeidins are a novel family of antiviral effectors against white spot syndrome virus (WSSV), which antagonize the envelope proteins to block viral entry. We found that the expression levels of four identified penaeidins from Litopenaeus vannamei, including BigPEN, PEN2, PEN3, and PEN4, were significantly induced in hemocytes during the early stage of WSSV infection. Knockdown of each penaeidin in vivo via RNA interference resulted in elevated viral loads and rendered shrimp more susceptible to WSSV, while the survival rate was rescued via the injection of recombinant penaeidins. All penaeidins, except PEN4, were shown to interact with several envelope proteins of WSSV, and all four penaeidins were observed to be located on the outer surface of the WSSV virion. Co-incubation of each recombinant penaeidin with WSSV inhibited virion internalization into hemocytes. More importantly, we found that PEN2 competitively bound to the envelope protein VP24 to release it from polymeric immunoglobulin receptor (pIgR), the cellular receptor required for WSSV infection. Moreover, we also demonstrated that BigPEN was able to bind to VP28 of WSSV, which disrupted the interaction between VP28 and Rab7the Rab GTPase that contributes to viral entry by binding with VP28. Taken together, our results demonstrated that penaeidins interact with the envelope proteins of WSSV to block multiple viral infection processes, thereby protecting the host against WSSV.

Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are vital signaling adaptor proteins for the innate immune response and are involved in many important pathways, such as the NF-κB-and interferon regulatory factor (IRF)-activated signaling pathways. In this study, the TRAF3 ortholog from the shrimp Litopenaeus vannamei (LvTRAF3) was cloned and characterized. LvTRAF3 has a transcript of 3,865 bp, with an open reading frame (ORF) of 1,002 bp and encodes a polypeptide of 333 amino acids, including a conserved TRAF-C domain. The expression of LvTRAF3 in the intestine and hemocyte was up-regulated in response to poly (I:C) challenge and white spot syndrome virus (WSSV) infection. RNAi knockdown of LvTRAF3 in vivo significantly increased WSSV gene transcription, viral loads, and mortality in WSSV-infected shrimp. Next, we found that LvTRAF3 was not able to induce the activation of the NF-κB pathway, which was crucial for synthesis of antimicrobial peptides (AMPs), which mediate antiviral immunity. Specifically, in dual-luciferase reporter assays, LvTRAF3 could not activate several types of promoters with NF-κB binding sites, including those from WSSV genes (wsv069, wsv056, and wsv403), Drosophila AMPs or shrimp AMPs. Accordingly, the mRNA levels of shrimp AMPs did not significantly change when TRAF3 was knocked down during WSSV infection. Instead, we found that LvTRAF3 signaled through the IRF-Vago antiviral cascade. LvTRAF3 functioned upstream of LvIRF to regulate the expression of LvVago4 and LvVago5 during WSSV infection in vivo. Taken together, these data provide experimental evidence of the participation of LvTRAF3 in the host defense to WSSV through the activation of the IRF-Vago pathway but not the NF-κB pathway.

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