Antiviral Immunity Of Shrimp Research Articles

Interferon-Induced Protein 6-16 (IFI6-16) from Litopenaeus vannamei Regulate Antiviral Immunity via Apoptosis-Related Genes.

A growing number of evidence shows that some invertebrates possess an antiviral immunity parallel to the interferon (IFN) system of higher vertebrates. For example, the IRF (interferon regulatory factor)–Vago–JAK/STAT regulatory axis in an arthropod, shrimp Litopenaeus vannamei (whiteleg shrimp) is functionally similar to the IRF–IFN–JAK/STAT axis of mammals. IFNs perform their cellular immunity by regulating the expression of target genes collectively referred to as IFN-stimulated genes (ISGs). However, the function of invertebrate ISGs in immune responses is almost completely unclear. In this study, a potential ISG gene homologous to the interferon-induced protein 6-16 (IFI6-16) was cloned and identified from L. vannamei, designated as LvIFI6-16. LvIFI6-16 contained a putative signal peptide in the N-terminal, and a classic IFI6-16-superfamily domain in the C-terminal that showed high conservation to other homologs in various species. The mRNA levels of LvIFI6-16 were significantly upregulated after the stimulation of poly (I:C) and challenges of white spot syndrome virus (WSSV). Moreover, silencing of LvIFI6-16 caused a higher mortality rate and heightened virus loads, suggesting that LvIFI6-16 could play a crucial role in defense against WSSV. Interestingly, we found that the transcription levels of several caspases were regulated by LvIFI6-16; meanwhile, the transcription level of LvIFI6-16 self was regulated by the JAK/STAT cascade, suggesting there could be a JAK/STAT–IFI6-16–caspase regulatory axis in shrimp. Taken together, we identified a crustacean IFI6-16 gene (LvIFI6-16) for the first time, and provided evidence that the IFI6-16 participated in antiviral immunity in shrimp.

Metabolomic Profiles in the Intestine of Shrimp Infected by White Spot Syndrome Virus and Antiviral Function of the Metabolite Linoleic Acid in Shrimp

White spot syndrome virus (WSSV) is a threatening pathogenic virus in shrimp culture, and at present, no effective strategy can prevent and control the disease. Intestinal flora and its metabolites are important for the resistance of shrimp to lethal pathogenic viruses. However, the changes of metabolites in the shrimp intestines after WSSV infection remain unclear. We established an artificial oral infection method to infect shrimp with WSSV and analyzed the metabolites in intestinal content of shrimp by HPLC and tandem mass spectrometry. A total of 78 different metabolites and five different metabolic pathways were identified. Among them, we found that the content of linoleic acid, an unsaturated fatty acid, increased significantly after WSSV infection, indicating that linoleic acid might be involved in antiviral immunity in shrimp. Further study showed that, after oral administration of linoleic acid, WSSV proliferation decreased evidently in the shrimp, and survival rate of the shrimp increased significantly. Mechanical analysis showed that linoleic acid directly bound to WSSV virions and inhibited the viral replication. Linoleic acid also promoted the expression of antimicrobial peptides and IFN-like gene Vago5 by activating the ERK-NF-κB signaling pathway. Our results indicated that WSSV infection caused metabolomic transformation of intestinal microbiota and that the metabolite linoleic acid participated in the immune response against WSSV in shrimp.

RPS27, a sORF-Encoded Polypeptide, Functions Antivirally by Activating the NF-κB Pathway and Interacting With Viral Envelope Proteins in Shrimp.

A small open reading frame (smORF) or short open reading frame (sORF) encodes a polypeptide of <100 amino acids in eukaryotes (50 amino acids in prokaryotes). Studies have shown that several sORF-encoded peptides (SEPs) have important physiological functions in different organisms. Many ribosomal proteins belonging to SEPs play important roles in several cellular processes, such as DNA damage repair and apoptosis. Several studies have implicated SEPs in response to infection and innate immunity, but the mechanisms have been unclear for most of them. In this study, we identified a sORF-encoded ribosomal protein S27 (RPS27) in Marsupenaeus japonicus. The expression of MjRPS27 was significantly upregulated in shrimp infected with white spot syndrome virus (WSSV). After knockdown of MjRPS27 by RNA interference, WSSV replication increased significantly. Conversely, after MjRPS27 overexpression, WSSV replication decreased in shrimp and the survival rate of the shrimp increased significantly. These results suggested that MjRPS27 inhibited viral replication. Further study showed that, after MjRPS27 knockdown, the mRNA expression level of MjDorsal, MjRelish, and antimicrobial peptides (AMPs) decreased, and the nuclear translocation of MjDorsal and MjRelish into the nucleus also decreased. These findings indicated that MjRPS27 might activate the NF-κB pathway and regulate the expression of AMPs in shrimp after WSSV challenge, thereby inhibiting viral replication. We also found that MjRPS27 interacted with WSSV's envelope proteins, including VP19, VP24, and VP28, suggesting that MjRPS27 may inhibit WSSV proliferation by preventing virion assembly in shrimp. This study was the first to elucidate the function of the ribosomal protein MjRPS27 in the antiviral immunity of shrimp.

Identification of Functional Gene Modules Associated With STAT-Mediated Antiviral Responses to White Spot Syndrome Virus in Shrimp.

White spot syndrome virus (WSSV) is one of the major threats to shrimp aquaculture. It has been found that the signal transducer and activator of transcription (STAT) protein plays an important role in the antiviral immunity of shrimp with a WSSV infection. However, the mechanism that underlies the STAT-mediated antiviral responses in shrimp, against WSSV infection, remains unclear. In this work, based on the gene expression profiles of shrimp with an injection of WSSV and STAT double strand RNA (dsRNA), we constructed a gene co-expression network for shrimp and identified the gene modules that are possibly responsible for STAT-mediated antiviral responses. These gene modules are found enriched in the regulation of the viral process, JAK-STAT cascade and the regulation of immune effector process pathways. The gene modules identified here provide insights into the molecular mechanism that underlies the STAT-mediated antiviral response of shrimp, against WSSV.

The Role of Cytokine PF4 in the Antiviral Immune Response of Shrimp.

During viral infection in vertebrates, cytokines play important roles in the host defense against the virus. However, the function of cytokines in invertebrates has not been well characterized. In this study, shrimp cytokines involved in viral infection were screened using a cytokine antibody microarray. The results showed that three cytokines, the Fas receptor (Fas), platelet factor 4 (PF4) and interleukin-22 (IL-22), were significantly upregulated in the white spot syndrome virus (WSSV)-challenged shrimp, suggesting that these cytokines played positive regulatory roles in the immune response of shrimp against the virus. Further experiments revealed that PF4 had positive effects on the antiviral immunity of shrimp by enhancing the shrimp phagocytic activity and inhibiting the apoptotic activity of virus-infected hemocytes. Therefore, our study presented a novel mechanism of cytokines in the innate immunity of invertebrates.

Shrimp with knockdown of LvSOCS2, a negative feedback loop regulator of JAK/STAT pathway in Litopenaeus vannamei, exhibit enhanced resistance against WSSV

JAK/STAT pathway is one of cytokine signaling pathways and mediates diversity immune responses to protect host from viral infection. In this study, LvSOCS2, a member of suppressor of cytokine signaling (SOCS) families, has been cloned and identified from Litopenaeus vannamei. The full length of LvSOCS2 is 1601 bp, including an 1194 bp open reading frame (ORF) coding for a putative protein of 397 amino acids with a calculated molecular weight of ∼42.3 kDa. LvSOCS2 expression was most abundant in gills and could respond to the challenge of LPS, Vibrio parahaemolyticus, Staphhylococcus aureus, Poly (I: C) and white spot syndrome virus (WSSV). There are several STAT binding motifs presented in the proximal promoter region of LvSOCS2 and its expression was induced by LvJAK or LvSTAT protein in a dose dependent manner, suggesting LvSOCS2 could be the transcriptional target gene of JAK/STAT pathway. Moreover, the transcription of DmVir-1, a read out of the activation of JAK/STAT pathway in Drosophila, was promoted by LvJAK but inhibited by LvSOCS2, indicating that LvSOCS2 could be a negative regulator in this pathway and thus can form a negative feedback loop. Our previous study indicated that shrimp JAK/STAT pathway played a positive role against WSSV. In this study, RNAi-mediated knockdown of LvSOCS2 shrimps showed lower susceptibility to WSSV infection and caused lessened virus loads, which further demonstrated that the JAK/STAT pathway could function as an anti-viral immunity in shrimp.

Virus-derived small RNAs in the penaeid shrimp Fenneropenaeus chinensis during acute infection of the DNA virus WSSV.

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are two classes of small RNAs (sRNAs) that are critical for virus-host interplay via the RNA interference (RNAi) pathway. One virus-derived siRNA and numerous miRNAs has been reported for the double-stranded DNA virus white spot syndrome virus (WSSV), however, the expression profiles of these different types of sRNAs have not been assessed. Here, by sequencing the sRNAs and mRNAs of WSSV-infected Chinese shrimp (Fenneropenaeus chinensis), we found that the viral transcripts were universally targeted by WSSV-derived siRNAs, supporting a pivotal role for RNAi in the anti-viral immunity of shrimp. The genesis of WSSV-derived siRNAs was associated with long RNA structures. Moreover, by separating miRNAs from siRNAs, 12 WSSV miRNAs were identified. Investigation of conserved viral miRNA targets in different host species indicated the involvement of viral miRNAs in host immune responses. Collectively, our data provide new insights into the role of the RNAi pathway in the interplay between DNA viruses and crustaceans.

A white spot syndrome virus microRNA promotes the virus infection by targeting the host STAT.

JAK/STAT pathway plays an important role in invertebrates during virus infection. However the microRNA (miRNA)-mediated regulation of JAK/STAT is not intensively investigated. Viral miRNAs, encoded by virus genome, have emerged as important regulators in the virus-host interactions. In this study, a WSSV (white spot syndrome virus)-encoded miRNA (WSSV-miR-22) was characterized in shrimp during virus infection. The results showed that the viral miRNA could promote WSSV infection in shrimp by targeting the host STAT gene. When the expression of JAK or STAT was knocked down by sequence-specific siRNA, the WSSV copies in shrimp were significantly increased, indicating that the JAK/STAT played positive roles in the antiviral immunity of shrimp. The further findings revealed that TEP1 and TEP2 were the effectors of JAK-STAT signaling pathway. The silencing of TEP1 or TEP2 led to an increase of WSSV copies in shrimp, showing TEP1 and TEP2 were involved in the shrimp immune response against virus infection. Therefore our study presented a novel viral miRNA-mediated JAK/STAT-TEP1/TEP2 signaling pathway in virus infection.

Function of shrimp STAT during WSSV infection

JAK/STAT signaling pathway plays key roles in the antiviral immunity of mammals, fish and insect. However, limited knowledge is known about the function of JAK/STAT signaling pathway in the antiviral immunity of shrimp although virus disease has caused severe mortality in shrimp aquaculture. In order to understand the function of JAK/STAT signaling pathway in the antiviral immunity of shrimp, dsRNA interfering technique was used to silence the expression of STAT gene in Litopenaeus vannamei, and the mortality of shrimp was detected after WSSV infection. Furthermore, the expressions of some potential target genes regulated by STAT or genes related to RNA interfering pathway were detected in STAT silenced shrimp during WSSV infection. The WSSV copy number in STAT silenced shrimp was 102–103 copies/ng DNA which was much lower than that in the control. The mortality in STAT silenced shrimp caused by WSSV infection decreased very significantly compared to their controls. The function of STAT was verified in vitro cultured cells of hematopoietic tissue of crayfish Cherax quadricarinatus by adding specific inhibitor of STAT3(S3I-201), and the cultured cells treated with S3I-201 showed much less WSSV copy number than their controls, which further suggested that STAT might be helpful for the replication of WSSV. Expression analysis on the potential STAT target genes and genes in RNA interfering pathway provide important information for understanding the functional mechanism of STAT in antiviral immunity of shrimp.

Nucleic acid-induced antiviral immunity in shrimp

Vertebrates detect viral infection predominantly by sensing viral nucleic acids to produce type I interferon (IFN). In invertebrates, it has been believed that the IFN system is absent and RNA interference is a sequence-specific antiviral pathway. In this study, we found that injection of nucleic acid mimics poly(I:C), poly(C:G), CL097, poly C and CpG-DNA, afforded shrimp antiviral immunity, which is similar to the vertebrate IFN system. Using suppression subtractive hybridization (SSH) method, 480 expression sequence tags were identified to be involved in the poly(I:C)-induced antiviral immunity of the model crustacean Litopenaeus vannamei, and 41% of them were new genes. In the SSH libraries, several IFN system-related genes such as dsRNA-dependent protein kinase PKR, Toll-like receptor 3 (TLR3) and IFNγ-inducible protein 30 were identified. L. vannamei IKKε, whose vertebrate homologs are central regulators of the IFN-producing pathway, could significantly activate IFN reporter genes in HEK293T cells. In crustacean databases, many genes homologous to genes of the vertebrate IFN response, such as IRFs, PKR, ADAR (adenosine deaminase, RNA-specific) and other interferon-stimulated genes (ISGs) were discovered. These results suggest that shrimp may possess nucleic acid-induced antiviral immunity.

Characterization of shrimp Drosha in virus infection

RNA interference (RNAi) mediated by microRNA (miRNA) is an evolutionarily conserved mechanism of posttranscriptional gene regulation in all eukaryotes, involving in natural antiviral immunity. The RNAase III Drosha is a key component for miRNA maturation. To date, however, the roles of Drosha in virus infection remain to be addressed. In this study, the Drosha was characterized in Marsupenaeus japonicus shrimp. The sequence analysis revealed that the shrimp Drosha gene encoded a 1081-amino-acid peptide, which comprised two tandem ribonuclease III C terminal domains and a double-stranded RNA binding motif. The shrimp Drosha was homologous with those of other animal species. The quantitative RT-PCR analysis revealed that the Drosha gene was highly expressed in lymphoid organ and was significantly up-regulated in response to WSSV challenge, suggesting that the Drosha was involved in the antiviral immunity of shrimp. The results showed that the knock down of Drosha gene led to the defect of miRNA maturation, and subsequent higher virus loads in shrimp. Our study presented that Drosha played important roles in the antiviral defense of shrimp.

Lack of evidence for Litopenaeus vannamei Toll receptor (lToll) involvement in activation of sequence-independent antiviral immunity in shrimp

Injection of non-specific dsRNA initiates a broad-spectrum innate antiviral immune response in the Pacific white shrimp, Litopenaeus vannamei, however, the receptor involved in recognition of this by-product of viral infections remains unknown. In vertebrates, dsRNA sensing is mediated by a class of Toll-like receptors (TLRs) and results in activation of the interferon system. Because a TLR (lToll) was recently characterized in L. vannamei, we investigated its potential role in dsRNA recognition. We showed that injection of non-specific RNA duplexes did not modify lToll gene expression. A reverse genetic approach was therefore implemented to study its role in vivo. Silencing of lToll did not impair the ability of non-specific dsRNA to trigger protection from white spot syndrome virus and did not increase the shrimp susceptibility to viral infection, when compared to controls. In contrast, gene-specific dsRNA injected to specifically silence lToll expression activated an antiviral response. These data strongly suggest that shrimp lToll plays no role in dsRNA-induced antiviral immunity.

YHV-protease dsRNA inhibits YHV replication in Penaeus monodon and prevents mortality

Yellow head virus infects cultured shrimps and causes severe mortality resulting in a great economic loss. Haemolymph injection of dsRNA(pro) corresponding to the protease motif of YHV genome resulted in a complete inhibition of YHV replication. The effect of dsRNA lasted for at least 5 days. Injecting sequence-unrelated dsRNA(gfp) or dsRNA(TSV-pol) also resulted in an inhibition of YHV replication but at a comparatively much less extent. Shrimp mortality was monitored for 10 days when more than 90% shrimps receiving no dsRNA died within 8 dpi. However, those receiving dsRNA(pro) showed no mortality. A partial mortality was observed among the shrimps receiving dsRNA(gfp) or dsRNA(TSV-pol). Thus, Penaeus monodon possesses the sequence-specific protection to YHV infection, most likely through the RNAi pathway, in addition to sequence-independent protection. It gives a new notion that dsRNA induction of antiviral immunity in shrimp goes through two pathways, sequence-independent and sequence-dependent.

SiRNA injection induces sequence-independent protection in Penaeus monodon against white spot syndrome virus

White spot syndrome virus (WSSV) is a major disease in crustaceans, particularly shrimp, due to the current intensity of aquaculture practices. Novel strategies including vaccination to control this virus would be highly desirable. However, invertebrates lack a true adaptive immune response system and seem to rely on various innate immune responses. An alternative and more specific approach to counteract WSSV infections in shrimp could be by the exploitation of RNA interference. As long dsRNA molecules induce a general, sequence-independent anti-viral immunity in shrimp [Robalino, J., Browdy, C.L., Prior, S., Metz, A., Parnell, P., Gross, P., Warr, G., 2004. J. Virol. 78, 10442–10448], it was investigated whether shorter 21 nt siRNAs with homology to the WSSV vp15 and vp28 genes would give a sequence-specific interference response in the shrimp Penaeus monodon. Vp28 siRNAs as well as nonspecific control gfp siRNAs were able to specifically and efficiently silence their homologous genes in a heterologous baculovirus insect cell expression system. However, in shrimps no such a specific effect was observed. Shrimp injected with vp15 or vp28 siRNAs before WSSV challenge gave a significantly lower mortality rate, but not significantly different when shrimps were injected with gfp siRNA. Thus, large dsRNA molecules as well as siRNAs induce a sequence-independent anti-viral immunity when injected in shrimp.