Effects of PmDOME and PmSTAT knockdown on white spot syndrome virus infection in Penaeus monodon.

Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway plays an important role in antiviral immunity. This research reports the full-length DOME receptor gene in Penaeus monodon (PmDOME) and examines the effects of PmDOME and PmSTAT silencing on immune-related gene expressions in shrimp hemocytes during white spot syndrome virus (WSSV) infection. PmDOME and PmSTAT were up-regulated in shrimp hemocytes upon WSSV infection. Suppression of PmDOME and PmSTAT showed significant impacts on the expression levels of ProPO2 (melanization), Vago5 (interferon-like protein) and several antimicrobial peptides, including ALFPm3, Penaeidin3, CrustinPm1 and CrustinPm7. Silencing of PmDOME and PmSTAT reduced WSSV copy numbers and delayed the cumulative mortality caused by WSSV. We postulated that suppression of the JAK/STAT signaling pathway may activate the proPO, IFN-like antiviral cytokine and AMP production, resulting in a delay of WSSV-related mortality.

White spot syndrome virus impact on the expression of immune genes and gut microbiome of black tiger shrimp Penaeus monodon.

The gut microbiome plays an essential role in the immune system of invertebrates and vertebrates. Pre and pro-biotics could enhance the shrimp immune system by increasing the phenoloxidase (PO), prophenoloxidase (ProPO), and superoxide dismutase activities. During viral infection, the host immune system alteration could influence the gut microbiome composition and probably lead to other pathogenic infections. Since the JAK/STAT pathway is involved in white spot syndrome virus (WSSV) infection, we investigated the intestine immune genes of STAT-silenced shrimp. During WSSV infection, expression levels of PmVago1, PmDoral, and PmSpätzle in PmSTAT-silenced shrimp were higher than normal. In addition, the transcription levels of antimicrobial peptides, including crustinPm1, crustinPm7, and PmPEN3, were higher in WSSV-challenged PmSTAT-silenced shrimp than the WSSV-infected normal shrimp. Meanwhile, PmSTAT silencing suppressed PmProPO1, PmProPO2, and PmPPAE1 expressions during WSSV infection. The microbiota from four shrimp tested groups (control group, WSSV-infected, PmSTAT-silenced, and PmSTAT-silenced infected by WSSV) was significantly different, with decreasing richness and diversity due to WSSV infection. The relative abundance of Bacteroidetes, Actinobacteria, and Planctomycetes was reduced in WSSV-challenged shrimp. However, at the species level, P. damselae, a pathogen to human and marine animals, significantly increased in WSSV-challenged shrimp. In constrast, Shewanella algae, a shrimp probiotic, was decreased in WSSV groups. In addition, the microbiota structure between control and PmSTAT-silenced shrimp was significantly different, suggesting the importance of STAT to maintain the homeostasis interaction with the microbiota.

PmAP2-ß depletion enhanced activation of the Toll signaling pathway during yellow head virus infection in the black tiger shrimp Penaeus monodon.

Yellow head virus (YHV) is a pathogen which causes high mortality in penaeid shrimp. Previous studies suggested that YHV enters shrimp cells via clathrin-mediated endocytosis. This research investigated the roles of clathrin adaptor protein 2 subunit ß (AP-2ß) from Penaeus monodon during YHV infection. PmAP2-ß was continuously up-regulated more than twofold during 6-36 hpi. Suppression of PmAP2-ß significantly reduced YHV copy numbers and delayed shrimp mortality. Quantitative RT-PCR revealed that knockdown of PmAP2-ß significantly enhanced the expression level of PmSpätzle, a signaling ligand in the Toll pathway, by 30-fold at 6 and 12 hpi. Moreover, the expression levels of gene components in the Imd and JAK/STAT signaling pathways under the suppression of PmAP2-ß during YHV infection were also investigated. Interestingly, anti-lipopolysaccharide factor isoform 3 (ALFPm3) was up-regulated by 40-fold in PmAP2-ß knockdown shrimp upon YHV infection. In addition, silencing of PmAP2-ß dramatically enhanced crustinPm1 expression in YHV-infected shrimp. Knockdown of ALFPm3 and crustinPm1 significantly reduced shrimp survival rate. Taken together, this work suggested that PmAP2-ß-deficiency promoted the Toll pathway signalings, resulting in elevated levels of ALFPm3 and crustinPm1, the crucial antimicrobial peptides in defence against YHV.

Role of Clathrin Assembly Protein-2 Beta Subunit during White Spot Syndrome Virus Infection in Black Tiger Shrimp Penaeus monodon.

White spot syndrome virus (WSSV) is one of the most lethal viruses severely affecting shrimp industry. This disease can cause 100% mortality of farmed shrimp within a week. This work aims to characterize clathrin assembly proteins in Penaeus monodon and investigate their roles in WSSV entry. In general, clathrin assembly proteins form complexes with specific receptors and clathrins, leading to clathrin-mediated endocytosis. Adaptor protein 2 (AP-2), which is responsible for endocytosis at plasma membrane, consists of four subunits including α, ß2, µ2 and σ2. Knockdown of clathrin coat AP17, or σ subunit of AP-2 dramatically reduced WSSV infectivity. Similar results were observed, when shrimp were pre-treated with chlorpromazine (CPZ), an inhibitor of clathrin-dependent endocytosis. The complete open reading frames of AP-2ß and µ subunits of P. monodon are reported. PmAP-2 ß was up-regulated about 4-fold at 6 and 36 h post-WSSV infection. Knockdown of PmAP-2ß delayed shrimp mortality during WSSV infection, of which WSSV intermediate early 1 gene expression was also down-regulated. Immunogold-labelling and transmission electron microscopy revealed that PmAP-2ß co-localized with WSSV particles at plasma membrane. In addition, PmAP-2ß-silencing significantly affected the expression levels of PmSTAT, PmDOME, PmDorsal and ALFPm3 during WSSV infection. It is possible that PmAP-2ß is associated with the JAK/STAT and the Toll pathway.

Molecular Characterization of Viral Responsive Protein 15 and Its Possible Role in Nuclear Export of Virus in Black Tiger Shrimp Penaeus monodon.

A viral responsive protein 15 from Penaeus monodon (PmVRP15) has been reported to be important for white spot syndrome virus (WSSV) infection in vivo. This work aims to characterize PmVRP15 and investigate its possible role in nuclear import/export of the virus. Circular dichroism spectra showed that PmVRP15 contains high helical contents (82%). Analytical ultracentrifugation suggested that PmVRP15 could possibly form oligomers in solution. A subcellular fractionation study showed that PmVRP15 was found in heavy and light membrane fractions, indicating that PmVRP15 may be associated with endoplasmic reticulum. Double-stranded RNAi-mediated knockdown of PmVRP15 gene expression in vitro showed no effect on WSSV copy number in whole hemocyte cells. However, PmVRP15 silencing resulted in an accumulation of WSSV DNA in the nucleus of PmVRP15-silenced hemocytes. Immunofluorescence confocal microscopy showed that PmVRP15 knockdown hemocytes had a much lower level of VP28 (WSSV envelope protein), in comparison to that in the control. It is likely that PmVRP15 may play a role in viral nuclear egress.