Roles of qseC mutation in bacterial resistance against anti-lipopolysaccharide factor isoform 3 (ALFPm3).

Propelled by global climate changes, the shrimp industry has been facing tremendous losses in production due to various disease outbreaks, particularly early mortality syndrome (EMS), a disease caused by Vibrio parahaemolyticus AHPND. Not only is the use of antibiotics as EMS control agents not yet been proven successful, but the overuse and misuse of antibiotics could also worsen one of the most challenging global health issues-antimicrobial resistance. To circumvent antibiotic usage, anti-lipopolysaccharide factor isoform 3 (ALFPm3), an antimicrobial peptide (AMP) derived from the shrimp innate immune system, was proposed as an antibiotic alternative for EMS control. However, prolonged use of AMPs could also lead to bacterial cross resistance with life-saving antibiotics used in human diseases. Here, we showed that ALFPm3-resistant strains of E. coli could be induced in vitro. Genome analysis of the resistant mutants revealed multiple mutations, with the most interesting being a qseC(L299R). A study of antibiotic susceptibility profile showed that the resistant strains harboring the qseC(L299R) not only exhibited higher degree of resistance towards polymyxin antibiotics, but also produced higher biofilm under ALFPm3 stress. Lastly, a single cell death analysis revealed that, at early-log phase when biofilm is scarce, the resistant strains were less affected by ALFPm3 treatment, suggesting additional mechanisms by which qseC orchestrates to protect the bacteria from ALFPm3. Altogether, this study uncovers involvement of qseC mutation in mechanism of resistance of the bacteria against ALFPm3 paving a way for future studies on sustainable use of ALFPm3 as an EMS control agent.

DnaJC16, the molecular chaperone, is implicated in hemocyte apoptosis and facilitates of WSSV infection in shrimp.

Chaperone proteins, including heat shock proteins (HSPs) and DnaJ proteins, are highly conserved and well known for their quick responses to environmental stresses and pathogen infections, especially viruses. However, how DnaJ, an HSP family member, in Penaeus vannamei responds to viral invasion has not been reported. In this research, the novel DnaJ homolog subfamily C member 16-like, or DnaJC16, was characterized in P. vannamei. It contains the DnaJ and thioredoxin domains. Phylogenetic tree analysis demonstrated the conservation of DnaJC16 among penaeid shrimp, where PvDnaJC16 was found to be closely related to DnaJC16 from Fenneropenaeus chinensis and Marsupenaeus japonicus. The transcripts of PvDnaJC16 were expressed in all the tissues tested, and the highest expression was in the lymphoid organs. As hemocytes are major immune tissue, we found significant upregulation of PvDnaJC16 in shrimp hemocytes after white spot syndrome virus (WSSV) infection. Furthermore, the suppression of PvDnaJC16 expression by RNA interference in WSSV-infected shrimp showed a decrease in replication and WSSV copy number. Interestingly, a dramatically high cumulative survival rate following the WSSV challenge (over 60%) was observed in PvDnaJC16-silenced shrimp. Meanwhile, the total hemocyte number was significantly increased in PvDnaJC16 knockdown. In addition, the expression of caspase-3 was reduced, as was the caspase-3/7 activity in PvDnaJC16 silencing. Additionally, the percentage of late apoptotic hemocytes diminished after PvDnaJC16 reduction, whereas the percentage of hemocyte viability increased. Our data reflect the fact that the upregulation of PvDnaJC16 expression upon WSSV infection enhances hemocyte apoptosis, which can accelerate viral spreading in shrimp.

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.

Prx4 acts as DAMP in shrimp, enhancing bacterial resistance via the toll pathway and prophenoloxidase activation.

Peroxiredoxin (Prx), an antioxidant enzyme family, has been identified as immune modulating damage-associated molecular patterns (DAMPs) in mammals but not in shrimp. Acute non-lethal heat shock (NLHS) that enhances shrimp Penaeus vannamei resistance to Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VPAHPND). Among the five P. vannamei Prxs (LvPrx) isoforms, LvPrx4, the most abundant in unchallenged shrimp hemocytes that was upregulated in hemocytes following NLHS treatment, is of great interest. The escalation of the LvPrx4 monomer in hemolymph of NLHS treated shrimp indicates that it probably acts as DAMP. This study revealed that pre-challenge with rLvPrx4 could prolong VPAHPND-infected shrimp survival, increase prophenoloxidase (proPO) activity and promote Toll pathway-related genes expression mediated by Toll-like receptor (TLR) 1 and 2. The presented findings elucidated the molecular mechanism of LvPrx4 monomer as DAMP in NLHS-induced VPAHPND resistance by inducing the TLR1/2 signaling pathway and the proPO activating system.

The interferon-like proteins, Vagos, in Fenneropenaeus merguiensis elicit antimicrobial responses against WSSV and VPAHPND infection.

The Vago interferon-like protein participates in the interplay between interferon regulatory factors and the expression of immune-responsive genes. Vago was initially perceived to participate only in the antiviral activation through JAK/STAT pathway. However, certain isoforms of Vago can stimulate antimicrobial responses. Here we identify Vago isoforms in Fenneropenaeus merguiensis (FmVagos) and how they function in antiviral and antibacterial responses against highly invasive pathogens, including white spot syndrome virus (WSSV) and Vibrio parahaemolyticus (VPAHPND). Three isoforms of FmVagos were identified: FmVago4, FmVago5a, and FmVago5b, and expressed throughout tissues of the shrimp. During infection, FmVago4, FmVago5a, and FmVago5b, were up-regulated after WSSV and VPAHPND challenges at certain time points. Pre-injection of purified recombinant FmVago4 (rVago4), FmVago5a (rVago5a), and FmVago5b (rVago5b) proteins could significantly reduce the mortality of shrimp upon WSSV infection, while the increase of survival rate of VPAHPND-infected shrimp was observed only in rVago4 treatment. The immunity routes that FmVagos might instigate in response to the pathogens were examined by qRT-PCR, revealing that the JAK/STAT pathway was activated after introducing rVago4, rVago5a, and rVago5b, while the Toll/IMD pathway and proPO system, combined with PO activity, were provoked only in the rVago4-treated shrimp. Our finding suggests cross-talk between Vago's antiviral and antimicrobial responses in shrimp immunity. These findings complement previous studies in which Vago and its specific isoform could promote viral and bacterial clearance in shrimp.

Shrimp pmo-miR-750 regulates the expression of sarcoplasmic calcium-binding protein facilitating virus infection in Penaeus monodon.

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and play crucial roles in antiviral responses. Penaeus monodon miR-750 (pmo-miR-750) was found to be strongly up-regulated in the late phase of white spot syndrome virus (WSSV) infection, but its function remains uncharacterized. Herein, the targets that were translationally down-regulated in the shrimp stomach following a pmo-miR-750 mimic injection were identified using two-dimensional gel electrophoresis. Sarcoplasmic calcium-binding protein (Scp) and actin1 (Act1) were revealed to be down-regulated protein spots. The genuine binding of pmo-miR-750 mimic to Scp but not Act1 mRNA was validated in vitro. In addition, a negative correlation between the Scp transcript and pmo-miR-750 expression level in WSSV-infected P. monodon stomach implies that pmo-miR-750 regulates Scp expression in vivo. When injected into WSSV-infected shrimp, the pmo-miR-750 mimic suppressed Scp expression but significantly increased the WSSV copy number. Consistent with the miRNA mimic-mediated Scp suppression, the loss of function assay of Scp in WSSV-challenged shrimp by RNA interference revealed a decreased survival rate with a dramatic increase in viral copy number. Besides that, apoptosis was activated in the hemocytes of the Scp knockdown shrimp upon WSSV infection. Collectively, our findings reveal that up-regulated pmo-miR-750 suppresses Scp expression at both the transcript and protein levels in the late stage of WSSV infection, which contributes to modulating apoptosis and eventually enabling viral propagation.

Transcriptome profiling reveals the novel immunometabolism-related genes against WSSV infection from Fenneropenaeus merguiensis.

The white spot syndrome virus (WSSV) has been considered a serious threat to shrimp aquaculture. Besides, the activation of cell metabolism as an immune reaction to the virus is now recognized as a piece of the pivotal puzzle of the antiviral responses. Hence, this study explores the relationship between metabolic gene expression and antiviral responses in shrimp using transcriptome analysis. The RNA-seq libraries of Fenneropenaeus merguensis hemocytes after WSSV challenge at early (6 hpi) and late (24 hpi) stages of infection were analyzed to identify differentially expressed genes (DEGs) that the WSSV subverted the expression. One-hundred-thirty-three DEGs that were expressed in response to WSSV infection at both stages were identified. Based on the GO annotation, they were related to innate immunity and metabolic pathway. The expression correlation between "full term" (NGS) and qRT-PCR of 16 representative DEGs is shown. Noticeably, the expression profiles of all the selected metabolic genes involved in glucose metabolism, lipid metabolism, amino acid metabolism, and nucleotide metabolism showed a specific correlation between NGS and qRT-PCR upon WSSV infection. Of these, we further characterized the function related to the WSSV response of glutamine: fructose-6-phosphate aminotransferase (FmGFAT), the rate-limiting enzyme of the hexosamine biosynthesis pathway, which was found to be up-regulated at the late stage of WSSV infection. Suppression of FmGFAT by RNA interference resulted in postponing the death of WSSV-infected shrimp and reduction of viral copy number. These results suggested that the FmGFAT is linked between metabolic change and WSSV responses in shrimp, where the virus-induced metabolic rewiring hijack biological compounds and/or energy sources to benefit the viral replication process.

Shrimp Vago5 activates an innate immune defense upon bacterial infection.

Acute hepatopancreatic necrosis disease, AHPND, caused by a specific Vibrio parahaemolyticus (VPAHPND) strain, results in a great loss of global shrimp production. This study performed suppression subtractive hybridization (SSH) to identify differentially expressed genes from white shrimp Penaeus vannamei hemocyte upon VPAHPND infection. Among the immune-related genes identified, Vago5, kunitz, secretory leukocyte proteinase inhibitor, and profilin are the most abundant genes classified as the up-regulated genes in the SSH library. The qRT-PCR results show that only Vago5 was highly up-regulated at 3 and 6 h post-VPAHPND challenge, whereas kunitz, secretory leukocyte proteinase inhibitor, and profilin were highly up-regulated at 48 h post-VPAHPND challenge. As an early VPAHPND infection-responsive gene, Vago5 was further functional characterized by RNA interference. Knockdown of Vago5 gene resulted in the significantly rapid increase of shrimp mortality and the number of bacteria in the stomach and hepatopancreas upon VPAHPND infection. Moreover, downstream genes of Toll, IMD, and JAK/STAT pathways and phenoloxidase system were analyzed for the expression in the VPAHPND-infected shrimp hemocyte after dsVago5 treatment. Vago5 gene knockdown resulted in a significant decrease in transcript levels of PEN4, TNF, and PO2 genes as well as PO activity in the hemolymph, suggesting that Vago5 might modulate antibacterial infection through activation of the genes in the NF-κB mediated pathways, JAK/STAT pathway, and phenoloxidase system.

Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor.

Acute hepatopancreatic necrosis disease (AHPND) caused by PirABVP-producing strain of Vibrio parahaemolyticus, VPAHPND, has seriously impacted the shrimp production. Although the VPAHPND toxin is known as the VPAHPND virulence factor, a receptor that mediates its action has not been identified. An in-house transcriptome of Litopenaeus vannamei hemocytes allows us to identify two proteins from the aminopeptidase N family, LvAPN1 and LvAPN2, the proteins of which in insect are known to be receptors for Cry toxin. The membrane-bound APN, LvAPN1, was characterized to determine if it was a VPAHPND toxin receptor. The increased expression of LvAPN1 was found in hemocytes, stomach, and hepatopancreas after the shrimp were challenged with either VPAHPND or the partially purified VPAHPND toxin. LvAPN1 knockdown reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VPAHPND bacteria in the stomach after VPAHPND toxin challenge. In addition, LvAPN1 silencing prevented the toxin from causing severe damage to the hemocytes and sustained both the total hemocyte count (THC) and the percentage of living hemocytes. We found that the rLvAPN1 directly bound to both rPirAVP and rPirBVP toxins, supporting the notion that silencing of LvAPN1 prevented the VPAHPND toxin from passing through the cell membrane of hemocytes. We concluded that the LvAPN1 was involved in AHPND pathogenesis and acted as a VPAHPND toxin receptor mediating the toxin penetration into hemocytes. Besides, this was the first report on the toxic effect of VPAHPND toxin on hemocytes other than the known target tissues, hepatopancreas and stomach.

Regulation of shrimp prophenoloxidase activating system by lva-miR-4850 during bacterial infection.

MicroRNAs (miRNAs) suppress gene expression and regulate biological processes. Following small RNA sequencing, shrimp hemocytes miRNAs differentially expressed in response to acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus (VPAHPND) were discovered and some were confirmed by qRT-PCR. VPAHPND-responsive miRNAs were predicted to target several genes in various immune pathways. Among them, lva-miR-4850 is of interest because its predicted target mRNAs are two important genes of the proPO system; proPO2 (PO2) and proPO activating factor 2 (PPAF2). The expression of lva-miR-4850 was significantly decreased after VPAHPND infection, whereas those of the target mRNAs, PO2 and PPAF2, and PO activity were significantly upregulated. Introducing the lva-miR-4850 mimic into VPAHPND-infected shrimps caused a reduction in the PO2 and PPAF2 transcript levels and the PO activity, but significantly increased the number of bacteria in the VPAHPND targeted tissues. This result inferred that lva-miR-4850 plays a crucial role in regulating the proPO system via suppressing expression of PPAF2 and PO2. To fight against VPAHPND infection, shrimp downregulated lva-miR-4850 expression resulted in proPO activation.

MicroRNA and mRNA interactions coordinate the immune response in non-lethal heat stressed Litopenaeus vannamei against AHPND-causing Vibrio parahaemolyticus.

While Vibrio parahaemolyticus (VPAHPND) has been identified as the cause of early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp, mechanisms of host response remain unknown. Understanding these processes is important to improve farming practices because this understanding will help to develop methods to enhance shrimp immunity. Pre-treatment of shrimp with 5-minute chronic non-lethal heat stress (NLHS) for 7 days was found to significantly increase Litopenaeus vannamei survival against VPAHPND infection. To elucidate the mechanism involved, mRNA and miRNA expression profiles from the hemocyte of L. vannamei challenged with VPAHPND after NLHS with corresponding control conditions were determined by RNA-Seq. A total of 2,664 mRNAs and 41 miRNAs were differentially expressed after the NLHS treatment and VPAHPND challenge. A miRNA-mRNA regulatory network of differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) was subsequently constructed and the interactions of DEMs in regulating the NLHS-induced immune-related pathways were identified. Transcriptomic data revealed that miRNA and mRNA interactions contribute to the modulation of NLHS-induced immune responses, such as the prophenoloxidase-activating system, hemocyte homeostasis, and antimicrobial peptide production, and these responses enhance VPAHPND resistance in L. vannamei.

Genome organization and definition of the Penaeus monodon viral responsive protein 15 (PmVRP15) promoter.

The viral responsive protein 15 from the black tiger shrimp Penaeus monodon (PmVRP15) is a highly responsive gene upon white spot syndrome virus (WSSV) challenge. It is identified from hemocyte and important for WSSV trafficking and assembly. However, the knowledge of PmVRP15 gene regulation is limited. In the present study, the genome organization and 5'upstream promoter sequences of PmVRP15 gene were investigated. The PmVRP15 gene was found to contain 4 exons interrupted by 3 introns and the start codon was located in the exon 2. The transcription start site and TATA box were also determined from the 5' upstream sequence. By using the narrow down experiment, the 5' upstream promoter active region was determined to be at the nucleotide positions -525 to +612. Mutagenesis of the putative transcription factor (TF) binding sites revealed that the binding site of interferon regulatory factor (IRF) (-495/-479) was a repressor-binding site whereas those of the octamer transcription factor 1 (Oct-1) (-275/-268) and the nuclear factor of activated T-cells transcription factor (NFAT) (-228/-223) were activator-binding sites. This is the first report on the transcription factors that might play essential roles in modulating the PmVRP15 gene expression. Nevertheless, the underlying regulation mechanism of PmVRP15 gene expression needs further investigation.

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.

Differentially expressed genes in hemocytes of Litopenaeus vannamei challenged with Vibrio parahaemolyticus AHPND (VPAHPND) and VPAHPND toxin.

While toxin-harboring Vibrio parahaemolyticus has been previously established as the causative agent of early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp, information on the mechanistic processes that happen in the host during infection is still lacking. Here, we examined the expression responses of the shrimp hemocyte transcriptome to V. parahaemolyticus AHPND (VPAHPND) by RNA sequencing (RNA-seq). Using libraries (SRA accession number SRP137285) prepared from shrimp hemocytes under experimental conditions, a reference library was de novo assembled for gene expression analysis of VPAHPND-challenged samples at 0, 3/6, and 48 h post infection (hpi). Using the library from 0-hpi as the control, 359 transcripts were found to be differentially expressed in the 3/6-hpi library, while 429 were differentially expressed in the 48-hpi library. The expression patterns reported in the RNA-seq of 9 representative genes such as anti-lipopolysaccharide factor (LvALF), crustin p (CRU), serpin 3 (SER), C-type lectin 3 (CTL), clottable protein 2 (CLO), mitogen-activated protein kinase kinase 4 (MKK4), P38 mitogen-activated protein kinase (P38), protein kinase A regulatory subunit 1 (PKA) and DNAJ homolog subfamily C member 1-like (DNJ) were validated by qRT-PCR. The expression of these genes was also analyzed in shrimp that were injected with the partially purified VPAHPND toxin. A VPAHPND toxin-responsive gene, LvALF was identified, and its function was characterized by RNA interference. LvALF knockdown resulted in significantly rapid increase of shrimp mortality caused by toxin injection. Protein-protein interaction analysis by molecular docking suggested that LvALF possibly neutralizes VPAHPND toxin through its LPS-binding domain. The data generated in this study provide preliminary insights into the differences in the immune response of shrimp to the bacterial and toxic aspect of VPAHPND as a disease.

Hemocyanin of Litopenaeus vannamei agglutinates Vibrio parahaemolyticus AHPND (VPAHPND) and neutralizes its toxin.

Acute hepatopancreatic necrosis disease, AHPND, caused by a specific strain of Vibrio parahaemolyticus (VPAHPND), results in great loss of global shrimp production. Despite this, studies on shrimp defense mechanisms protecting against AHPND are few. In this study, suppression subtractive hybridization (SSH) was performed to identify differentially expressed genes from white shrimp Litopenaeus vannamei hepatopancreas upon VPAHPND infection at the early stages: 3 and 6 h post challenge and in the late stage at 48 h post challenge. Hemocyanin (HMC) is the most abundant gene identified as the up-regulated gene in the SSH library. Various hemocyanin subunits such as hemocyanin (HMC), hemocyanin subunit L1 (HMCL1), L2 (HMCL2), L3 (HMCL3), and L4 (HMCL4) were analyzed for their expression levels upon VPAHPND infection and in response to challenge with partially purified toxin of VPAHPND by qRT-PCR. Only HMC was highly up-regulated at 3 and 6 h post challenge in response to VPAHPND challenge. Two HMC subunits, HMCL3 and HMCL4, were up-regulated in the early phase of VPAHPND toxin injection. Furthermore, all subunits were down-regulated in the late phase of VPAHPND and toxin challenges. The native hemocyanin protein purified from shrimp hemolymph, identified as mixture of HMC and HMCL1, exhibited agglutination activity on VPAHPND. Injecting the purified native hemocyanin along with VPAHPND into shrimp decreased the number of bacteria in the hemolymph as compared to the VPAHPND challenged control. Moreover, pre-incubation of the purified native hemocyanin and VPAHPND toxin prior to injection into shrimp resulted in the decrease of cumulative mortality of shrimp when compared to the control. In addition, protein-protein interaction analysis carried out by ELISA technique indicated that hemocyanin exhibited VPAHPND toxin-neutralizing activity through direct interaction with PirA subunit with a dissociation constant of 6.83 × 10-6 M. Our results indicated that upon VPAHPND infection the expression of hemocyanin was induced and hemocyanin functions might involve agglutination of invading VPAHPND and also neutralization of VPAHPND secreted toxin via direct interacting with the PirA protein.

WSSV-responsive gene expression under the influence of PmVRP15 suppression.

The viral responsive protein 15 from black tiger shrimp Penaeus monodon (PmVRP15), is highly up-regulated and produced in the hemocytes of shrimp with white spot syndrome virus (WSSV) infection. To investigate the differential expression of genes from P. monodon hemocytes that are involved in WSSV infection under the influence of PmVRP15 expression, suppression subtractive hybridization (SSH) of PmVRP15-silenced shrimp infected with WSSV was performed. The 189 cDNA clones of the forward library were generated by subtracting the cDNAs from WSSV-infected and PmVRP15 knockdown shrimp with cDNAs from WSSV-infected and GFP knockdown shrimp. For the opposite subtraction, the 176 cDNA clones in the reverse library was an alternative set of genes in WSSV-infected shrimp hemocytes in the presence of PmVRP15 expression. The abundant genes in forward SSH library had a defense/homeostasis of 26%, energy/metabolism of 23% and in the reverse SSH library a hypothetical protein with unknown function was found (30%). The differential expressed immune-related genes from each library were selected for expression analysis using qRT-PCR. All selected genes from the forward library showed high up-regulation in the WSSV-challenged PmVRP15 knockdown group as expected. Interestingly, PmHHAP, a hemocyte homeostasis associated protein, and granulin-like protein, a conserved growth factor, are extremely up-regulated in the absence of PmVRP15 expression in WSSV-infected shrimp. Only transcript level of transglutaminase II, that functions in regulating hematopoietic tissue differentiation and inhibits mature hemocyte production in shrimp, was obviously down-regulated as observed from SSH results. Taken together, our results suggest that PmVRP15 might have a function relevant to hemocyte homeostasis during WSSV infection.

A Pacifastacus leniusculus serine protease interacts with WSSV.

Serine proteases are involved in many critical physiological processes including virus spread and replication. In the present study, we identified a new clip-domain serine protease (PlcSP) in the crayfish Pacifastacus leniusculus hemocytes, which can interact with the White Spot Syndrome Virus (WSSV) envelope protein VP28. It was characterized by a classic clip domain with six strictly conserved Cys residues, and contained the conserved His-Asp-Ser (H-D-S) motif in the catalytic domain. Furthermore, signal peptide prediction revealed that it has a 16-residue secretion signal peptide. Tissue distribution showed that it was mainly located in P. leniusculus hemocytes, and its expression was increased in hemocytes upon WSSV challenge. In vitro knock down of PlcSP decreased both the expression of VP28 and the WSSV copy number in hematopoietic stem (HPT) cells. Accordingly, these data suggest that the new serine protease may be of importance for WSSV infection into hematopoietic cells.

Antiviral action of the antimicrobial peptide ALFPm3 from Penaeus monodon against white spot syndrome virus.

The anti-lipopolysaccharide factor isoform 3 (ALFPm3), the antimicrobial peptide from Penaeus monodon, possesses antibacterial and antiviral activities. Although the mechanism of action of ALFPm3 against bacteria has been revealed but its antiviral mechanism is still unclear. To further study how the ALFPm3 exhibits antiviral activity against the enveloped virus, white spot syndrome virus (WSSV), the ALFPm3-interacting proteins from WSSV were sought and identified five ALFPm3-interacting proteins, WSSV186, WSSV189, WSSV395, WSSV458, and WSSV471. Only the interaction between ALFPm3 and WSSV189, however, has been confirmed to be involved in anti-WSSV activity of ALFPm3. Herein, the interactions between ALFPm3 and rWSSV186, rWSSV395, rWSSV458, or rWSSV471 were further analyzed and confirmed by in vitro pull-down assay. Western blot analysis and immunoelectron microscopy showed that the uncharacterized proteins, WSSV186 and WSSV471, were nucleocapsid and envelope proteins, respectively. The decrease of shrimp survival after injection the shrimp with mixtures of each rWSSV protein, rALFPm3 and WSSV as compared to those injected with rALFPm3-neutralizing WSSV was clearly observed indicating that all rWSSV proteins could interfere with the neutralization effect of rALFPm3 on WSSV similar to that reported previously for WSSV189. Morphological change on WSSV after incubation with rALFPm3 was observed by TEM. The lysed WSSV virions were clearly observed where both viral envelope and nucleocapsid were dismantled. The results lead to the conclusion that the ALFPm3 displays direct effect on the viral structural proteins resulting in destabilization and breaking up of WSSV virions.

WSV399, a viral tegument protein, interacts with the shrimp protein PmVRP15 to facilitate viral trafficking and assembly.

Viral responsive protein 15 (PmVRP15) has been identified as a highly up-regulated gene in the hemocyte of white spot syndrome virus (WSSV)-infected shrimp Penaeus monodon. However, the function of PmVRP15 in host-viral interaction was still unclear. To elucidate PmVRP15 function, the interacting partner of PmVRP15 from WSSV was screened by yeast two-hybrid assay and then confirmed by co-immunoprecipitation (Co-IP). Only WSV399 protein was identified as a PmVRP15 binding protein; however, the function of WSV399 has not been characterized. Localization of WSV399 on the WSSV virion was revealed by immunoblotting analysis (in vitro) and immunoelectron microscopy (in vivo). The results showed that WSV399 is a structural protein of the WSSV virion and is particularly located on the tegument. Gene silencing of wsv399 in WSSV-infected shrimp reduced the percentage of cumulative mortality by 74%, although the expression level of a viral replication marker gene, vp28, was not changed suggesting that WSV399 might not involved in viral replication but viral assembly. Because it has already been known that tegument proteins function in capsid transport during viral trafficking and assembly, interaction between PmVRP15 on hemocyte nuclear membrane and the WSV399 viral tegument protein suggests that PmVRP15 might be required for trafficking and assembly of WSSV during infection.