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Virus infection causes the metabolic disorder of host cells, whereas the metabolic disorder of cells is one of the major causes of tumorigenesis, suggesting that antiviral molecules might possess anti-tumor activities by regulating cell metabolism. As the key regulators of gene expression, long non-coding RNAs (lncRNAs) play vital roles in the regulation of cell metabolism. However, the influence of antiviral lncRNAs on tumorigenesis has not been explored. To address this issue, the antiviral and anti-tumor capacities of shrimp lncRNAs were characterized in this study. The results revealed that shrimp lncRNA06, having antiviral activity in shrimp, could suppress the tumorigenesis of human gastric cancer stem cells (GCSCs) via triggering apoptosis of GCSCs in a cross-species manner. Shrimp lncRNA06 could sponge human miR-17-5p to suppress the stemness of GCSCs via the miR-17-5p-p21 axis. At the same time, shrimp lncRNA06 could bind to ATP synthase subunit beta (ATP5F1B) to enhance the stability of the ATP5F1B protein in GCSCs, thus suppressing the tumorigenesis of GCSCs. The in vivo data demonstrated that shrimp lncRNA06 promoted apoptosis and inhibited the stemness of GCSCs through interactions with ATP5F1B and miR-17-5p, leading to the suppression of the tumorigenesis of GCSCs. Therefore, our findings highlighted that antiviral lncRNAs possessed anti-tumor capacities and that antiviral lncRNAs could be the anti-tumor reservoir for the treatment of human cancers.

In invertebrates, hemocytes are the key factors in innate immunity. However, the types of invertebrate immune hemocytes are unclassified due to the limitation of morphological classification. To determine the immune hemocytes of crustaceans, the heterogeneity of hemocytes of shrimp Marsupenaeus japonicus and crayfish Procambarus clarkii , two representative crustacean species, were characterized in this study. The results of single-cell RNA sequencing indicated that shrimp and crayfish contained 11 and 12 types of hemocytes, respectively. Each of different types of hemocytes specifically expressed the potential marker genes. Based on the responses of shrimp and crayfish to the infection of white spot syndrome virus (WSSV) and the challenge of lipopolysaccharide (LPS), four types of immune hemocytes of crustaceans were classified, including semi-granular hemocytes involved in antimicrobial peptide production, granular hemocytes responsible for the production of antimicrobial peptides, hemocytes related to cell proliferation and hemocytes in immunity-activated state. Therefore, our study provided the first classification of crustacean hemocytes as well as of immune hemocytes of crustaceans at the single-cell resolution, which would be helpful to understand the innate immunity of invertebrates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Xin and Zhang.)

As a kind of important economic marine crustaceans in aquaculture, shrimp can be infected by more than 20 viruses. To fight against the virus invasion, shrimp have developed the innate immunity, including RNA interference (RNAi). RNAi, mediated by short interfering RNAs (siRNAs) or microRNAs (miRNAs), plays important roles in virus-host interactions. At present, RNAi is considered to be an efficient antiviral response of shrimp. The siRNA-based RNAi, first recognized as an antiviral response of animals to resist RNA viruses, has emerged in animals as an efficient antiviral strategy against the invasion of DNA viruses and RNA viruses. In shrimp, as well as in other animals, siRNA contains a seed region (2nd-7th nt) and a supplementary region (12th-17th nt). Based on the findings in shrimp and other animals, miRNAs are essential regulators of virus-host interactions, such as virus infection/latency, and host apoptosis, autophagy and phagocytosis. Except for the seed sequence (2nd-7th), the complementary bases (to the target mRNA sequence) of a miRNA 9th-18th non-seed sequence are essential for the miRNA targeting. So far, rapidly growing evidences have supported the existence of functional RNAi machinery in shrimp. In this review, we summarize the progress of RNAi in the antiviral immune response of shrimp. The potential applications of RNAi to control shrimp diseases were also summarized., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The elevated NH 3 -N and NO 2 -N pollution problems in mariculture have raised concerns because they pose threats to animal health and coastal and offshore environments. Supplement of Marichromatium gracile YL28 (YL28) into polluted shrimp rearing water and sediment significantly decreased ammonia and nitrite concentrations, showing that YL28 functioned as a novel safe marine probiotic in the shrimp culture industry. The diversity of aquatic bacteria in the shrimp mariculture ecosystems was studied by sequencing the V4 region of 16S rRNA genes, with respect to additions of YL28 at the low and high concentrations. It was revealed by 16S rRNA sequencing analysis that Proteobacteria, Planctomycete and Bacteroidetes dominated the community (>80% of operational taxonomic units (OTUs)). Up to 41.6% of the predominant bacterial members were placed in the classes Gammaproteobacteria (14%), Deltaproteobacteria (14%), Planctomycetacia (8%) and Alphaproteobacteria (5.6%) while 40% of OTUs belonged to unclassified ones or others, indicating that the considerable bacterial populations were novel in our shrimp mariculture. Bacterial communities were similar between YL28 supplements and control groups (without addition of YL28) revealed by the β-diversity using PCoA, demonstrating that the additions of YL28 did not disturb the microbiota in shrimp mariculture ecosystems. Instead, the addition of YL28 increased the relative abundance of ammonia-oxidizing and denitrifying bacteria. The quantitative PCR analysis further showed that key genes including nifH and amoA involved in nitrification and nitrate or nitrite reduction significantly increased with YL28 supplementation ( p < 0.05). The supplement of YL28 decreased the relative abundance of potential pathogen Vibrio . Together, our studies showed that supplement of YL28 improved the water quality by increasing the relative abundance of ammonia-oxidizing and denitrifying bacteria while the microbial community structure persisted in shrimp mariculture ecosystems.

Synonymous single nucleotide polymorphisms (SNPs) are involved in codon usage preference or mRNA splicing. Up to date, however, the role of synonymous SNPs in immunity remains unclear. To address this issue, the SNPs of white spot syndrome virus (WSSV) were characterized in shrimp in the present study. Our results indicated that there existed synonymous SNPs in the mRNAs of wsv151 and wsv226, two viral genes of WSSV. In the presence of SNP siRNA, wild-type siRNA, wild-type mRNA and SNP mRNA of wsv151 or wsv226, RNAi was significantly suppressed, showing that the synonymous SNPs of wsv151 and wsv226 played negative roles in host siRNA pathway due to mismatch of siRNA with its target. In insect cells, the mismatch, caused by synonymous SNPs of wsv151 or wsv226, between siRNA and its target inhibited the host RNAi. Furthermore, the data revealed that the co-injection of SNP siRNA and wild-type siRNA of wsv151 or wsv226 into WSSV-infected shrimp led to a significant increase of WSSV copies compared with that of SNP siRNA alone or wild-type siRNA alone, indicating that the synonymous SNPs of viral genes could be a strategy of virus escaping host siRNA pathway in shrimp in vivo . Therefore, our study provided novel insights into the underlying mechanism of virus escaping host antiviral RNAi immunity by synonymous SNPs of viral genes.

A tumor can be regarded as a cell mass with a biologic hierarchy that is orchestrated by cancer stem cells. The specific features of cancer stem cells may result from a series of gene expression regulatory mechanisms. As important regulatory factors of gene expression, microRNAs (miRNAs) have critical roles in the self-renewal, pluripotency, differentiation, and tumorigenicity of cancer stem cells. However, the effects of animal miRNAs on cancer stem cells have not been investigated extensively. Here, we report that miRNA of a shrimp, Marsupenaeus japonicus (mja-miR-35-3p), which possesses antiviral activity in shrimp, could suppress the proliferation of melanoma and breast cancer stem cells, but not cancer nonstem cells, by arresting the cell cycle in the G 1 phase and inducing apoptosis. Shrimp mja-miR-35-3p (named mja-miR-35) targets the human peptidylprolyl cis / trans isomerase, never in mitosis gene a-interacting 1 ( PIN1 ) gene, which is up-regulated in cancer stem cells. The results indicated that PIN1 silencing caused cell cycle arrest in the G 1 phase, induced apoptosis, and decreased the sphere-forming capacity of cancer stem cells, but not cancer nonstem cells, showing that PIN1 had beneficial effects against the stemness of cancer stem cells. The in vivo data revealed that shrimp mja-miR-35 suppressed the stemness of cancer stem cells in mice by inhibiting the PIN1 antiapoptotic-cell cycle pathway. These findings demonstrated that the miRNAs of aquatic animals might be an important source for discovering human antitumor drugs.-Zhang, S., Zhang, X. Shrimp miRNA suppresses the stemness of human cancer stem cells via the PIN1 pathway.

White shrimp Litopenaeus vannamei are widely cultured in the world and white spot syndrome virus (WSSV) led to huge economic losses in the shrimp industry every year. In the present study, miRNAs involved in the response of shrimp L. vannamei to WSSV infection were obtained through the Illumina HiSeq 2500 high-throughput next-generation sequencing technique. A total number of 7 known miRNAs and 54 putative novel miRNAs were obtained. Among them, 14 DEMs were identified in the shrimp infected with WSSV. The putative target genes of these DEMs were related to host immune response or signaling pathways, indicating the importance of miRNAs in shrimp against WSSV infection. The results will provide information for further research on shrimp response to virus infection and contribute to the development of new strategies for effective protection against WSSV infections., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The DCP1-DCP2 complex can regulate the antiviral immunity of animals by the decapping of retrovirus RNAs and the suppression of RNAi during RNA virus infection. However, the influence of DCP1-DCP2 complex on DNA virus infection and the regulation of DCP1-DCP2 complex by microRNAs (miRNAs) remain unclear. In this study, the role of miRNA-regulated DCP1-DCP2 complex in DNA virus infection was characterized. Our results showed that the DCP1-DCP2 complex played a positive role in the infection of white spot syndrome virus (WSSV), a DNA virus of shrimp. In the DCP1-DCP2 complex, the N-terminal regulatory domain of DCP2 was interacted with the EVH1 domain of DCP1. Furthermore, shrimp miRNA miR-87 inhibited WSSV infection by targeting the host DCP2 gene and viral miRNA WSSV-miR-N46 took a negative effect on WSSV replication by targeting the host DCP1 gene. Therefore, our study provided novel insights into the underlying mechanism of DCP1-DCP2 complex and its regulation by miRNAs in virus-host interactions. IMPORTANCE: During RNA virus infection, the DCP1-DCP2 complex can play important roles in the animal antiviral immunity by decapping retrovirus RNAs and suppressing RNAi. In the present study, the findings indicated that the silencing of DCP1 and DCP2 inhibited the infection of WSSV, a DNA virus of shrimp, suggesting that the DCP1-DCP2 complex facilitated DNA virus infection. Due to the suppressive role of the DCP1-DCP2 complex in shrimp RNAi against WSSV infection, the DCP1-DCP2 complex could promote WSSV infection in shrimp. The results showed that WSSV-miR-N46 and shrimp miR-87 could respectively suppress the expressions of DCP1 and DCP2 to affect virus infection. Therefore, our study contributed novel aspects of the DCP1-DCP2 complex and its regulation by miRNAs in virus-host interactions., (Copyright © 2019. Published by Elsevier Ltd.)

In vertebrates, pyroptosis is an intense inflammatory form of programmed cell death. This death pathway is critical for controlling pathogenic infection. In invertebrates, however, due to the lack of adaptive immune response, it is still elusive whether Caspase 1-dependent cell death pathway exists. In this study, our data showed that Caspase 1-mediated cell death was activated by white spot syndrome virus to counteract virus infection. Caspase 1 had a higher expression in hemocytes and lymphoid-like organ in shrimp and WSSV infection was promoted upon the inhibition of Caspase 1 enzymatic activity. IL-1β-like protein was identified as the substrate of Caspase 1 and its interaction with Caspase 1 was validated ectopically and endogenously. Moreover, IL-1β like protein was released into extracellular contents under WSSV infection and Prophenoloxidase system was activated, resulting in the reduction of WSSV. Our data unraveled a previously unidentified mechanism through which Caspase 1-dependent cell death controlled virus infection in shrimp., (Copyright © 2018 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) function as crucial suppressors of gene expression via translational repression or direct mRNA degradation. However, the mechanism of multi-gene regulation by a host miRNA in antiviral immunity has not been extensively explored. In this study, the regulation of two white spot syndrome virus (WSSV) genes by its host ( Marsupenaeus japonicus shrimp) miRNA (shrimp miR-1000) was characterized. The miRNA target gene prediction showed that only two virus genes ( wsv191 and wsv407 ) might be the targets of miR-1000. The results of insect cell transfection assays revealed that shrimp miR-1000 could target multiple virus genes ( wsv191 and wsv407 ). The mRNA degradation analysis and RNA FISH (fluorescence in situ hybridization) analysis indicated that miR-1000 triggered the mRNA degradation of target genes through 5'-3' exonucleolytic digestion in vivo and thereby inhibited the virus infection in shrimp. The miRNA-mediated 5'-3' exonucleolytic digestion of target mRNAs stopped near the 3'UTR (3'untranslated region) sequence complementary to the seed sequence of miR-1000. Therefore, our study provided novel insights into how a host miRNA targeted multiple viral genes and prevented host from virus infection.

Virus infection can change host's metabolism, while tumorigenesis results from metabolic disorder. MicroRNAs (miRNAs), crucial regulatory factors overlaying transcriptional control mechanisms, can guide metabolic homeostasis. In terms of metabolic disorder, antiviral miRNAs may have anti-tumor activity. However, this issue has not been extensively investigated. In the present study, the results revealed that shrimp mja-miR-35, which showed antiviral activity in shrimp against white spot syndrome virus (WSSV) infection, could suppress the metastasis of breast cancer by targeting human CHI3L1 gene of M2 macrophages in a cross-phylum manner. Furthermore, the feed expressing shrimp mja-miR-35 had antiviral capacity in shrimp and anti-tumor activity in humans, leading to the simultaneous control of virus infection and tumor progression. Therefore, our findings indicated that the antiviral miRNAs derived from shrimp stress responses against virus infection might be an important source of human anti-tumor drugs and miRNAs could bridge the control of aquaculture diseases and the prevention of human tumors.

Autophagy is known to be involved in viral infection. However, the relationship between autophagy and viral infection has not been characterized. MicroRNAs (miRNAs), important regulators of gene transcription, play central roles in many biologic processes, including autophagy and viral infection. It was therefore of interest to investigate the miRNAs regulating viral infection and host autophagy. In this study the viral infection (white spot syndrome virus, WSSV) correlated positively with the host autophagy in shrimp. Two host miRNAs (miR-71 and -13b) had significant effects on viral infection and host autophagy. MiR-71 regulated the viral infection and host autophagy by targeting the host calcification-associated peptide-1 ( cap-1 ) gene. MiR-13b targeted the host knickkopf gene to simultaneously regulate viral infection and host autophagy. When the expressions of miR-71 and -13b were silenced in shrimp, the autophagy could not be induced by WSSV infection and the autophagy induction did not influence the virus replication, indicating that miR-71 and -13b were necessary for the viral infection and host autophagy. In this context, the findings of this study revealed a novel mechanism of miRNAs bridging the viral infection and host autophagy in vivo -He, Y., Sun, Y., Zhang, X. Noncoding miRNAs bridge virus infection and host autophagy in shrimp in vivo., (© FASEB.)

In eukaryotes, microRNAs (miRNAs) serve as regulators of many biological processes, including virus infection. An miRNA can generally target diverse genes during virus-host interactions. However, the regulation of gene expression by multiple miRNAs has not yet been extensively explored during virus infection. This study found that the Spaztle (Spz)-Toll-Dorsal-antilipopolysaccharide factor (ALF) signaling pathway plays a very important role in antiviral immunity against invasion of white spot syndrome virus (WSSV) in shrimp ( Marsupenaeus japonicus ). Dorsal , the central gene in the Toll pathway, was targeted by two viral miRNAs (WSSV-miR-N13 and WSSV-miR-N23) during WSSV infection. The regulation of Dorsal expression by viral miRNAs suppressed the Spz-Toll-Dorsal-ALF signaling pathway in shrimp in vivo , leading to virus infection. Our study contributes novel insights into the viral miRNA-mediated Toll signaling pathway during the virus-host interaction. IMPORTANCE An miRNA can target diverse genes during virus-host interactions. However, the regulation of gene expression by multiple miRNAs during virus infection has not yet been extensively explored. The results of this study indicated that the shrimp Dorsal gene, the central gene in the Toll pathway, was targeted by two viral miRNAs during infection with white spot syndrome virus. Regulation of Dorsal expression by viral miRNAs suppressed the Spz-Toll-Dorsal-ALF signaling pathway in shrimp in vivo , leading to virus infection. Our study provides new insight into the viral miRNA-mediated Toll signaling pathway in virus-host interactions., (Copyright © 2017 American Society for Microbiology.)

RNAi, mediated by microRNAs (miRNAs), has attracted increasing attention for its important role in cross-talk between host and virus. However, the role of host miRNA in the virus infection in vivo has not been intensively investigated. In this study, the effects of a shrimp miRNA (miR-965) on the white spot syndrome virus (WSSV) infection were characterized. The results indicated that the expression of miR-965 was significantly upregulated in shrimp in response to the WSSV challenge, suggesting its involvement in the virus infection. The miR-965 silencing led to significant increases of WSSV copies and virus-infected shrimp mortality, while the miR-965 overexpression resulted in the decreased WSSV copies and virus-infected shrimp mortality, indicating that miR-965 played a negative role in the WSSV infection. The further data revealed that miR-965 inhibited the virus infection by targeting the viral wsv240 gene, an important gene required for the WSSV infection in shrimp. The results demonstrated that miR-965 could promote the shrimp phagocytosis against virus infection by targeting the shrimp ATG5 (autophagy related 5) gene. Therefore, our findings presented novel evidence to better understand the anfractuous host-virus interactions in vivo., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

The p53 protein plays an important role in apoptosis which is involved in the immunity of animals. However, effects of the miRNA-mediated regulation of p53 expression on apoptosis and virus infection are not extensively investigated. To address this issue, the miRNA-mediated p53-dependent apoptotic pathway was explored in this study. The results indicated that p53 could regulate the apoptotic activity of Marsupenaeus japonicas shrimp and influence the infection of white spot syndrome virus (WSSV). The further data presented that miR-1000 could target the 3'-untranslated region (3'UTR) of p53 gene. The results of in vivo experiments showed that the miR-1000 overexpression led to significant decreases of shrimp apoptotic activity and the capacity of WSSV infection, while the miR-1000 silencing resulted in significant increases of apoptotic activity and virus infection, indicating that miR-1000 took great effects on apoptosis and virus infection by targeting p53. Therefore, our study revealed a novel mechanism that the miR-1000-p53 pathway regulated apoptosis and virus infection in shrimp., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The process of programmed cell death process or apoptosis can be regulated by microRNAs, 21-25 nt short non-coding RNAs. However, a comprehensive view of apoptosis-associated miRNAs has not been intensively characterized. In this study, the shrimp miRNA microarray data showed that 199 miRNAs were involved in the regulation of apoptosis, among which 8 miRNAs were evolutionarily conserved in animals. The loss-of-function experiments in vivo in shrimp revealed that miR-100 was served as an anti-apoptosis miRNA through targeting the mRNA of trypsin gene. The results indicated that the silencing of miR-100 expression resulted in the increase of apoptotic activity of shrimp hemocytes and further led to the decreases of virus genome copies in shrimp and virus-infected shrimp mortality compared with the controls. The findings showed that miR-100-trypsin signaling pathway played an important role in the antiviral immunity by regulating apoptosis. Therefore, our study presented a novel miR-100-mediated pathway in the regulation of apoptosis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The culture of penaeid shrimp is rapidly developing as a major business endeavor worldwide. However, viral diseases have caused huge economic loss in penaeid shrimp culture industries. Knowledge of shrimp innate immunity and antiviral responses has made important progress in recent years, allowing the design of better strategies for the prevention and control of shrimp diseases. In this study, we have updated information on shrimp antiviral immunity and interactions between shrimp hosts and viral pathogens. Current knowledge and recent progress in immune signaling pathways (e.g., Toll/IMD-NF-κB and JAK-STAT signaling pathways), RNAi, phagocytosis, and apoptosis in shrimp antiviral immunity are discussed. The mechanism of viral infection in shrimp hosts and the interactions between viruses and shrimp innate immune systems are also analyzed., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Viruses, in particular DNA viruses, generate microRNAs (miRNAs) to control the expression of host and viral genes. Due to their essential roles in virus-host interactions, viral miRNAs have attracted extensive investigations in recent years. To date, however, most studies on viral miRNAs have been conducted in cell lines. In this study, the viral miRNAs from white spot syndrome virus (WSSV) were characterized in shrimp in vivo. On the basis of our previous study and small RNA sequencing in this study, a total of 89 putative WSSV miRNAs were identified. As revealed by miRNA microarray analysis and Northern blotting, the expression of viral miRNAs was tissue specific in vivo. The results indicated that the viral miRNA WSSV-miR-N24 could target the shrimp caspase 8 gene, and this miRNA further repressed the apoptosis of shrimp hemocytes in vivo. As a result, the number of WSSV copies in shrimp in vivo was significantly increased compared with the control level (WSSV only). Therefore, our study presents the first report on the in vivo molecular events of viral miRNA in antiviral apoptosis.

Viral microRNAs (miRNAs), most of which are characterized in cell lines, have been found to play important roles in the virus life cycle to avoid attack by the host immune system or to keep virus in the latency state. Viral miRNAs targeting virus genes can inhibit virus infection. In this study, in vivo findings in Marsupenaeus japonicus shrimp revealed that the viral miRNAs could target virus genes and further promote the virus infection. The results showed that white spot syndrome virus (WSSV)-encoded miRNAs WSSV-miR-66 and WSSV-miR-68 were transcribed at the early stage of WSSV infection. When the expression of WSSV-miR-66 and WSSV-miR-68 was silenced with sequence-specific anti-miRNA oligonucleotides (AMOs), the number of copies of WSSV and the WSSV-infected shrimp mortality were significantly decreased, indicating that the two viral miRNAs had a great effect on virus infection. It was revealed that the WSSV wsv094 and wsv177 genes were the targets of WSSV-miR-66 and that the wsv248 and wsv309 genes were the targets of WSSV-miR-68. The data demonstrate that the four target genes play negative roles in the WSSV infection. The targeting of the four virus genes by WSSV-miR-66 and WSSV-miR-68 led to the promotion of virus infection. Therefore, our in vivo findings show a novel aspect of viral miRNAs in virus-host interactions.

RNAi, a crucial pathway in animals to defend against virus infection, is mediated directly by RNA-induced silencing complex (RISC) in an ATP-dependent manner. The RISC comprises one strand of short interfering RNA (siRNA) and multiprotein including Argonaute protein, which can cleave target RNAs. However, the proteins interacted with siRNA are not extensively explored. In this study, an antiviral siRNA (vp28-siRNA) targeting the vp28 gene of shrimp white spot syndrome virus was characterized. Based on the biotin/streptavidin affinity screening, it was found that the shrimp arginine kinase was specifically bound with the vp28-siRNA. The co-immunoprecipitation assays revealed that the siRNA was directly interacted with arginine kinase, suggesting that arginine kinase was an essential component of RNA-induced silencing complex. Therefore, our study presented a novel finding on the RISC components, which would be helpful to reveal the molecular events in the RNAi pathway.

Growing evidence from mammals suggests that host microRNAs (miRNAs) play important roles in the antiviral immune response. However, the roles of invertebrate miRNAs in response to virus infection remain to be investigated. Based on our previous studies, the shrimp miR-7 was found to be upregulated in response to white spot syndrome virus (WSSV) infection. In this study, the results showed that shrimp miR-7 could target the 3'-untranslated region (3'UTR) of the WSSV early gene wsv477, implying that miR-7 was involved in viral DNA replication. In insect High Five cells, the synthesized miR-7 significantly decreased the expression level of the fluorescent construct bearing the 3'UTR of wsv477 compared with the expression of the control constructs. When the activity of transfected miR-7 was blocked by locked-nucleic-acid (LNA)-modified anti-miR-7 oligonucleotide (AMO-miR-7), the repression of luciferase gene expression by miR-7 was relieved. In vivo, when the synthesized miR-7 was injected into shrimp, the numbers of WSSV genome copies/mg gills were 1,000-fold lower than those of WSSV only at 72 and 96 h postinfection. The results indicated that the blocking of endogenous miR-7 by AMO-miR-7 led to about a 10-fold increase of WSSV genome copies/mg gills in WSSV-infected shrimp compared with the control WSSV only. Further, it was revealed that the host Dicer1 was an important component for the biogenesis of miR-7, which had a large effect on virus infection. Therefore, our study revealed a novel regulatory function for an invertebrate miRNA in host-virus interactions by targeting the viral early gene.

Phagocytosis, which is of fundamental importance for innate and adaptive immunity in animals, is driven by organization of the actin cytoskeleton. To date, however, the molecular events involved in the regulation of phagocytosis through reorganization of actin by small G proteins remains to be elucidated. To address this issue, the molecular mechanism of Rab6 in phagocytosis against virus infection in invertebrates was characterized in this study. The results showed that the Rab6 obtained from shrimp could interact with actin to regulate shrimp hemocyte phagocytosis through induction of the rearrangement of actin to protect against white spot syndrome virus (WSSV) infection. The Rab6 protein in Drosophila melanogaster shared the same mechanism of action as that of Rab6 in shrimp, indicating that the function of Rab6 in phagocytosis was conserved in invertebrates. By comparison with the early marker (Rab5) and late marker (LAMP1) of phagosomes, Rab6 was critically involved in the regulation of actin organization throughout the entire phagocytosis process. The presence of the evolutionarily conserved amino acid sequences of Rab6 in invertebrates and vertebrates indicated a conserved mechanism of Rab6 function in phagocytosis of animals. Therefore, our findings presented novel molecular events in the regulation of phagocytosis by small G proteins.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The accumulating evidence indicates that the viral structural proteins play critical roles in virus infection. However, the interaction between the viral structural protein and host cytoskeleton protein in virus infection remains to be addressed. In this study, the viral VP466 protein, one of the major structural proteins of shrimp white spot syndrome virus (WSSV), was characterized. The results showed that the suppression of VP466 gene expression led to the inhibition of WSSV infection in shrimp, indicating that the VP466 protein was required in virus invasion. It was found that the VP466 protein was interacted with the host cytoskeleton protein tropomyosin. As documented, the VP466-tropomyosin interaction facilitated the WSSV infection. Therefore our findings revealed a novel molecular mechanism in the virus invasion to its host, which would be helpful to better understand the molecular events in virus infection in invertebrate., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Widespread evidence indicates that the structural proteins of virus play very important roles in virus-host interactions. However, the effect of viral proteins on host immunity has not been addressed. Our previous studies revealed that the host shrimp Rab6 (termed as PjRab previously), tropomyosin, β-actin and the white spot syndrome virus (WSSV) envelope protein VP466 formed a complex. In this study, the VP466 protein was shown to be able to bind host Rab6 protein and increase its GTPase activity in vivo and vitro. Thus, VP466 could function as a GTPase-activating protein (GAP) of Rab6. In the VP466-Rab-actin pathway, the increase of the Rab6 activity induced rearrangements of the actin cytoskeleton, resulting in the formation of actin stress fibers which promoted the phagocytosis against virus. Therefore our findings revealed that a viral protein could be employed by host to initiate the host immunity, representing a novel molecular mechanism in the virus-host interaction. Our study would help to better understand the molecular events in immune response against virus infection in invertebrates., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Guided by miRNAs, RNAi plays an important role in virus-host interactions by fine-tuning gene expression. Many viral and cellular miRNAs are involved in virus infection, though no comprehensive general model for miRNAs derived from invertebrate DNA viruses exists for their function in eukaryotic systems, despite extensive research on miRNAs. To address this issue, the miRNAs from shrimp white spot syndrome virus (WSSV), a DNA virus with a 305 kb double-stranded circular DNA genome, were characterized. Based on WSSV miRNA microarray and northern blot analyses, WSSV was shown to possess the capacity to encode 40 distinct viral miRNAs, a miRNA content roughly 360 times greater than that of humans. These findings suggested that the high content of viral miRNAs might greatly contribute to viral variability in response selective pressures in the host environment. Transcription analysis revealed that 80% of WSSV miRNAs were expressed during early stages of viral infection, indicating their importance in initial infective processes. Additionally, biogenesis of viral miRNAs was demonstrated to be dependent on host Drosha and Dicer 1, mediated by Ago 1, and viral miRNAs, including WSSV-miR211 and WSSV-miR212, were required for successful WSSV infection. During WSSV infection, numerous viral genes were likely targeted by WSSV miRNAs. The current study presented the first comprehensive view of viral miRNAs encoded by an invertebrate DNA virus, providing insight into the molecular events of virus-host interactions.

Background: MicroRNAs (miRNAs) are key posttranscriptional regulators of gene expression that are implicated in many processes of eukaryotic cells. It is known that the expression profiles of host miRNAs can be reshaped by viruses. However, a systematic investigation of marine invertebrate miRNAs that respond to virus infection has not yet been performed., Results: In this study, the shrimp Marsupenaeus japonicus was challenged by white spot syndrome virus (WSSV). Small RNA sequencing of WSSV-infected shrimp at different time post-infection (0, 6, 24 and 48 h) identified 63 host miRNAs, 48 of which were conserved in other animals, representing 43 distinct families. Of the identified host miRNAs, 31 were differentially expressed in response to virus infection, of which 25 were up-regulated and six down-regulated. The results were confirmed by northern blots. The TargetScan and miRanda algorithms showed that most target genes of the differentially expressed miRNAs were related to immune responses. Gene ontology analysis revealed that immune signaling pathways were mediated by these miRNAs. Evolutionary analysis showed that three of them, miR-1, miR-7 and miR-34, are highly conserved in shrimp, fruit fly and humans and function in the similar pathways., Conclusions: Our study provides the first large-scale characterization of marine invertebrate miRNAs that respond to virus infection. This will help to reveal the molecular events involved in virus-host interactions mediated by miRNAs and their evolution in animals.

White spot syndrome virus (WSSV) is a major shrimp viral pathogen responsible for large economic losses to shrimp aquaculture all over the world. The RNAi mediated by siRNA contributes a new strategy to control this viral disease. However, the efficient approach to deliver the siRNA into shrimp remains to be addressed. In this investigation, an antiviral vp28-siRNA was encapsulated in β-1,3-D-glucan, and then the β-1,3-D-glucan-encapsulated vp28-siRNA particles (GeRPs) were delivered into Marsupenaeus japonicus shrimp. The results showed that the vp28-siRNA in GeRPs could be released in hemocytes of shrimp. It was found that the GeRPs containing the vp28-siRNA inhibited the replication of WSSV in vivo, which presented a better antiviral activity than the non-encapsulated vp28-siRNA. Further evidence indicated that the mortality of WSSV-infected shrimp was significantly delayed by the GeRPs containing vp28-siRNA. Therefore, our study presented that the glucan-encapsulated siRNA might represent a novel potential therapeutic or preventive approach to control the shrimp disease.

Argonaute (Ago) protein, the central component of the RNA interference (RNAi) pathway, plays important roles in host innate antiviral immunity. Most organisms harbor a large number of different Ago proteins and isoforms; however, the roles of Ago isoforms in immune defense against pathogens remain unclear. In the present study, three Argonaute-1 (Ago1) isoforms, termed Ago1A, Ago1B, and Ago1C, were found in Marsupenaeus japonicus shrimp. Quantitative real-time PCR (polymerase chain reaction) revealed that isoforms Ago1A and Ago1B containing an insertion sequence in the PIWI domain, were significantly up-regulated in lymphoid organ and hemolymph, and also upon white spot syndrome virus (WSSV) challenge, indicating the involvement of Ago1A and Ago1B in antiviral immunity. The results showed that silencing of Ago1A with a sequence-specific siRNA led to a significant increase of WSSV loads. It was revealed that knockdown of Ago1B mRNA by 37-70% resulted in higher virus loads in shrimp. However, upon silencing Ago1B by more than 85%, a two-fold increase in Ago1A mRNA was observed but viral load was the same as untreated controls challenged with WSSV, suggesting that the simultaneous up-regulation of Ago1A might compensate for the loss of Ago1B. These data indicated that Ago1A played more important roles in the antiviral immune response than Ago1B. The simultaneous inhibition of Ago1A and Ago1B resulted in a greater increase in viral loads than Ago1A or Ago1B alone, indicating that Ago1A and Ago1B isoforms were involved in shrimp antiviral immunity. It was revealed that Ago1C had no effect on virus infection. Therefore, the current study presented the first report on the contribution of Ago isoforms in the invertebrate defense against virus infection.

The global shrimp aquaculture has been consistently beset by diseases that cause severe losses in production. To fight various harmful pathogens, the enhanced shrimp immunity by immunostimulants would play key roles against the invading pathogens. In aquaculture, however, the target proteins/genes which can be used for the screening of immunostimulants are very limited. Based on our previous study, in the present study, the shrimp Ran protein, which was required in shrimp antiviral phagocytosis, was used as the target protein to screen for immunostimulants. The GTPase activity assays showed that the IL-4 and lysophosphatidylcholine molecules could enhance the activity of Ran protein, suggesting that the two molecules might function in phagocytosis. When the IL-4 and lysophosphatidylcholine were respectively injected into shrimp, the results indicated that the two molecules enhanced the hemocytic phagocytosis against white spot syndrome virus (WSSV), suggesting that they improved the activity of phagocytosis through the activation of the Ran protein. It was evidenced that the enhancement of phagocytosis activity effectively inhibited the WSSV infection in shrimp, which further led to the decrease of mortalities of WSSV-infected shrimp. Therefore, our study presented a novel strategy for the screening of immunostimulants by using the key proteins in immune responses of aquatic organisms as the target proteins, which would be very helpful for the development of efficient approaches to prevent the aquatic organisms from pathogen infections., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Ran, an abundant GTPase that is highly conserved in eukaryotes from yeast to human, has been implicated in many aspects of nuclear structure and function. Recently it is revealed that the Ran GTPase can regulate the hemocytic phagocytosis of shrimp by interaction with myosin. However the regulation of Ran gene expression remains unknown. In this study, the promoter of shrimp Ran gene was identified which contained a typical TATA box. The results showed that the shrimp Ran protein was bound with the Ran promoter in Drosophila S2 cells. Based on luciferase assays, our study indicated that the transcription of Ran gene could be regulated by the interaction between the Ran promoter and the Ran protein, suggesting the existence of a feedback regulation in Ran gene expression. Therefore our study presented a novel finding on the feedback regulation of gene transcription., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

Epidemic diseases cost large amount of economic loss in the shrimp aquaculture. To control the epidemic diseases, it is a very efficient approach to enhance the shrimp immunity by immunostimulants. In aquaculture, however, the applications of the available immunostimulants are very limited due to the lack of information about the roles of these immunostimulants in animal immunity. In the present study, a caspase protein (PjCaspase), required in shrimp antiviral apoptosis, was used as the target protein to screen for small molecules which would enhance the shrimp immunity. Based on screening using the EGFP-PjCaspase fusion protein in insect cells, four small molecules could enhance the activity of PjCaspase protein. Among them, IL-2 and evodiamine were further evidenced to enhance the apoptotic activity of shrimp hemocytes in vivo, suggesting that the small molecules improved the activity of apoptosis through the activation of the PjCaspase protein. The results indicated that the enhancement of apoptotic activity effectively inhibited the white spot syndrome virus (WSSV) infection in shrimp, which further led to the decrease of mortalities of WSSV-infected shrimp. Therefore, our study, for the first time, presented that the strategy using the key proteins in immune responses of aquatic organisms as the target proteins was a very efficient approach for the screening of immunostimulants to prevent the aquatic organisms from pathogen infections.

Vertebrates achieve adaptive immunity of all sorts against pathogens through the diversification of antibodies. However the mechanism of invertebrates' innate immune defense against various pathogens remains largely unknown. Our study used shrimp and white spot syndrome virus (WSSV) to show that PjCaspase, a caspase gene of shrimp that is crucial in apoptosis, possessed gene sequence diversity. At present, the role of gene sequence diversity in immunity has not been characterized. To address this issue, we compared the PjCaspase gene sequence diversities from WSSV-free and WSSV-resistant shrimp. The sequence analysis indicated that the PjCaspase gene from the WSSV-resistant shrimp contained a special fragment, designated as fragment 3 (221-229 aa). Down-regulation or overexpression of the PjCaspase gene containing fragment 3 led to significant inhibition or enhancement of virus-induced apoptosis, but had no effect on bacterium challenge. We found evidence that the silencing or overexpression of this gene led to a 7-fold increase or 11-fold decrease of WSSV copies, respectively. Our results suggested that the PjCaspase gene containing fragment 3 provided the molecular basis for the antiviral defense of shrimp. This study represented the first report of the role of gene sequence diversity in the immunity of an invertebrate against virus infection. Invertebrates may employ this gene sequence diversity as a system to avoid pathogen interference with their immune response.

Ran GTPases, one family of small G protein superfamily, have been widely demonstrated to be involved in the transport system between cytoplasm and the nucleus. However, the function of Ran GTPase in immunity remains unclear. In our study, it was found that the Ran GTPase (designated as PjRan) was up-regulated in virus-resistant shrimp, indicating that the PjRan might be implicated in the innate immune system against virus infection. On the basis of protein interactions, it was found that the PjRan interacted with myosin, a crucial protein in the process of phagocytosis to form a protein complex. The RNAi and mRNA assays showed that the PjRan could regulate shrimp hemocytic phagocytosis. Further data evidenced that the depletion of PjRan by RNAi caused a significant increase of virus copies, and the overexpression of PjRan resulted in a significant decrease of virus copies, suggesting that the PjRan participated in the antiviral immunity by regulating phagocytosis. Therefore, our study revealed a completely novel aspect of Ran GTPase in phagocytosis by the direct interaction with the cytoskeleton protein and presented a novel pathway concerning to antiviral immunity, which will help to better understand the molecular events in immune response against virus infection in invertebrates.

The antiviral effectiveness of three major immune responses including phagocytosis, apoptosis and proPO system in the shrimp Marsupenaeus japonicus was characterized. It was found that the suppression of apoptosis and phagocytosis in vivo by their corresponding inhibitors resulted in the increase of white spot syndrome virus (WSSV) copies and shrimp mortality, whereas the inhibition of phenol oxidase generated the least influence on WSSV infection and shrimp mortality. These results suggest that apoptosis and phagocytosis were the essential immune responses to protect shrimp from virus infection, while the phenol-oxidase-dependent proPO system plays a comparatively minor role in antiviral defense of shrimp.

Rab GTPases, members of the Ras superfamily, play important roles in phagosome formation and maturation. However, the involvement of Rab protein in phagocytosis against invading pathogens in crustacean remains unknown. In the present study, the RNAi and mRNA overexpression assays were conducted to elucidate the function of shrimp Rab gene (designated as PjRab) in hemocytic phagocytosis against bacterium. The results indicated that the phagocytic percentage and phagocytic index using FITC-labeled Vibrio parahemolyticus were significantly decreased when the PjRab gene was silenced by sequence-specific siRNA, suggesting that the PjRab protein was essential in hemocytic phagocytosis. On the other hand, the overexpression of PjRab gene leaded to the increase of phagocytic percentage and phagocytic index. The findings indicated that the PjRab protein was involved in the regulation of hemocytic phagocytosis of shrimp. Our report on the regulation of phagocytosis by Rab GTPase would contribute a better clue to realize the still poorly understood molecular events involved in shrimp as well as crustacean immune response.

Since it was proposed to be a tumor suppressor in 1991, the QM protein has attracted intensive studies in plants, animals and fungi. Up to date, however, the function of QM protein remains unknown. In this investigation, it was found that the shrimp QM gene (designated as PjQM) was significantly up-regulated in virus- resistant shrimp, suggesting that the PjQM was involved in shrimp immunity. The GST pull-down assays showed that the PjQM protein interacted with shrimp hemocyanin and myosin, indicating that the PjQM protein might participate in prophenoloxidation (proPO) activation system of shrimp immunity. As revealed by RNAi assays, it was demonstrated for the first time that the QM protein could regulate the activity of phenol oxidase, a key enzyme in the proPO activation system of invertebrate immunity. This discovery showed a novel aspect of QM protein in arthropod immune response, which contributed a better understanding to the still poorly understood molecular events involved in innate immunity of arthropods., ((c) 2008 S. Karger AG, Basel.)

Caspases are central effectors in apoptosis. In this investigation, a novel caspase gene (designated as PjCaspase) obtained from the marine shrimp Marsupenaeus japonicus was found to be significantly upregulated in survivors of WSSV-challenged shrimp, suggesting that it might be involved in shrimp antiviral immunity. As revealed by RNAi assays, when the PjCaspase gene was silenced by gene-specific siRNA, the WSSV-induced apoptosis was significantly inhibited. The results showed that the PjCaspase gene was essential in the virus-induced apoptosis of shrimp. Based on the quantitative PCR detection, it was shown that the PjCaspase gene silencing resulted in the increase of virus copies, indicating that apoptosis played a key role in antiviral process of shrimp. As well known, caspase-3 and -8 are crucial caspases in apoptosis. The discovery of PjCaspase in this study would contribute another essential caspase involved in apoptosis against virus infection, which might reveal an ancient mechanism of caspase activation in invertebrate immunity against viruses.

Diseases caused by viruses, especially white spot syndrome virus (WSSV), are the greatest challenge to worldwide shrimp aquaculture. Therefore, the innate immunity of shrimp has attracted extensive attentions these years. To date, however, no mechanism of immuno-related signal transduction pathway has been reported. In this investigation, an important signal transduction factor-Ran gene encoding ras-related nuclear protein (Ran protein) was characterized in shrimp. The shrimp Ran gene, without introns when compared with genomic DNA, was 645 bp in length. The GTP-binding assay showed that the Ran protein had GTP-binding activity. The results of RT-PCR and Western blot indicated that the transcript and protein of Ran were detected in every tissue of shrimp including hepatopancreas, haemolymph, gill, intestine, heart and muscle. In the WSSV-resistant and WSSV-infected shrimp at 4h postinfection, the Ran gene was obviously up-regulated, indicating that it played a role in shrimp immunity against virus infection. This study, therefore, might provide a clue to elucidate shrimp innate immunity.

The molecular mechanisms of the immune system against virus in shrimp are not well known, despite its economic importance as an aquaculture species. In this investigation, a Rab gene (named as PjRab gene) was obtained from Peneaus japonicus shrimp, which exhibited high homology with Rab 6 of other species. The PjRab protein, having GTP-binding activity, contained characteristic signatures of Rab proteins with 6 GTP binding domains and 5 Rab specific domains. However, the PjRab protein exhibited a very different prenylation site (CLLNL) at its C-terminus from most of other Rabs. The PjRab gene was ubiquitously expressed in shrimp tissues. Real-time PCR revealed that the PjRab gene was up-regulated in WSSV-resistant shrimp, suggesting that the PjRab protein might play an important role in shrimp immune response against virus infection. This discovery might contribute better understanding to the molecular events involved in shrimp as well as invertebrate immune responses.

White spot syndrome virus (WSSV) is a major shrimp pathogen causing large economic losses all over the world. So far, however, there is no efficient approach to control this virus. RNA interference (RNAi), which has been applied to silence virus genes in eukaryotic organisms. In this investigation, a specific 21bp short interfering RNA (vp28-siRNA) targeting a major envelope protein gene (vp28) of WSSV was used to induce gene silencing in vivo in Penaeus japonicus shrimp. It was found that the transcription and expression of vp28 gene were silenced by the sequence-specific vp28-siRNA. However, the RNAi effect disappeared or significantly weakened even if one-nucleotide mutation existed in the vp28-siRNA. As revealed by quantitative PCR, the vp28-siRNA caused a significant reduction in viral DNA production of WSSV-infected shrimp. When treated with the vp28-siRNA, WSSV-infected shrimp had a reduced mortality rate. After three injections of the vp28-siRNA, the virus was completely eradicated from WSSV-infected shrimp. These findings suggest that RNAi is capable of silencing sequence-specific genes of WSSV and might constitute a new therapeutic strategy for WSSV infection in shrimp.

In invertebrates, C-type lectin plays an important role in innate immunity by mediating the recognition of pathogens to host cells and clearing microinvaders. A few C-type lectins have been identified from shrimps, but none of their gene or protein sequences is known to date. In this paper, a C-type lectin (named PmLec) specific for bacterial lipopolysaccharide was purified from the serum of the shrimp Penaeus monodon. The binding of PmLec to lipopolysaccharide was mainly mediated through the O-antigen. PmLec had a strong hemagglutinating and bacterial-agglutinating activity as well as an opsonic effect that enhances hemocyte phagocytosis. The PmLec cDNA sequence was obtained from the cDNA library of P. monodon by polymerase chain reaction with the degenerated primer designed according to the amino-terminal residue sequence of purified PmLec. A 546-bp open reading frame was found to encode a putative protein comprising 182 amino acids and containing a preceding signal peptide of 17 amino acids. A C-type lectin domain existed in PmLec, but no glycosylation site was found. The recombinant PmLec protein expressed in Escherichia coli also showed the same agglutinating activity and opsonic effect as that of the native protein. This is the first report of a lectin cDNA from the shrimp. PmLec functions as a pattern-recognition protein and an opsonin in the shrimp, and it provides a clue to elucidate the role of lectin in the innate immunity of aquatic invertebrates at the molecular level.

White spot syndrome virus (WSSV) is a major shrimp pathogen causing large economic losses. In an attempt to identify the envelope proteins involved in virus infection, antisera against six WSSV envelope proteins were used in neutralization assays conducted in vivo. The results showed that the virus infection could be significantly delayed or neutralized by antibodies against three WSSV envelope proteins (VP68, VP281 and VP466). This neutralization was further confirmed by quantitative PCR. It could be concluded that the viral envelope proteins VP68, VP281 and VP466 played roles in WSSV infection to shrimp.

Penaeid shrimp aquaculture has suffered from many diseases, especially from viral origin such as white spot syndrome virus (WSSV). In an attempt to obtain antiviral-relevant proteins, two peptides with molecular masses at 73 and 75kDa were isolated from shrimp Penaeus monodon using affinity chromatography coupled with the purified WSSV or a fish iridovirus (Singapore grouper iridovirus, SGIV), and identified as hemocyanin by mass spectrometry. The results, using fish viruses capable of cell culture, showed for the first time that the hemocyanin had non-specific antiviral properties and no cytotoxicity against host cells.

Diseases caused by viruses especially by white spot syndrome virus (WSSV) are the greatest challenge to worldwide shrimp aquaculture. The innate immunity of shrimp has attracted extensive attention, but no factor involved in the virus resistance has been reported. Here we report for the first time the identification of an antiviral gene from shrimp Penaeus monodon. A differential cDNA (designated as PmAV) cloned from virus-resistant shrimp P. monodon by differential display (DD) was found to have an open reading frame (ORF) encoding a 170 amino acid peptide with a C-type lectin-like domain (CTLD). The PmAV gene was expressed in Escherichia coli and the protein was purified. Recombinant PmAV protein displayed a strong antiviral activity in inhibiting virus-induced cytopathic effect in fish cell in vitro. Moreover, native PmAV protein was isolated from shrimp hemolymph by immuno-affinity chromatography and confirmed by Western blot. No agglutination activity was observed both in recombinant and native PmAV protein. Immunohistological study showed that PmAV protein was located mainly in the cytoplasm, and not bound to the shrimp WSSV. It implies that the antiviral mechanism of PmAV protein is not by inhibiting the attachment of virus to target host cell. The discovery of PmAV gene might provide a clue to elucidate the innate immunity of marine invertebrates and would be helpful to shrimp viral disease control.