White Spot Syndrome Virus Proliferation Research Articles

Evaluation of the antiviral activity of chlorogenic acid against white spot syndrome virus

The rapid development of crustacean aquaculture is threatened by white spot syndrome virus (WSSV), which is one of the deadliest pathogens affecting various crustacean species. Based on antiviral plant screening, we found that chlorogenic acid (CGA), a polyphenolic component of Arctium lappa Linn., possessed strong activity against WSSV infection in a red swamp crayfish (Procambarus clarkii) model. The results revealed that CGA dose-dependently inhibited WSSV proliferation in the gill and haemocytes with the inhibition rates (50 mg/kg) were 94.7% and 95.03%, respectively. Through a serial of mechanical experiments, we found that CGA arrested WSSV replication by regulating innate immune defense to reduce viral gene transcription, inducing apoptosis to restrict viral spread, and enhancing anti-inflammatory and antioxidant activities to alleviate oxidative and inflammatory damage caused by WSSV. Significant improvements in survival of WSSV infected crayfish have been made through injection and dietary administration of CGA compared to control group. In a static cohabitation model, pre-incubation of CGA suppressed the horizontal transmission of WSSV between infected donor crayfish and health crayfish. Thus, we have identified CGA as a potent WSSV inhibitor with high potential for further development as prophylaxis and therapy agents against WSSV outbreak in crustacean aquaculture.

KCMF1-like suppresses white spot syndrome virus infection by promoting apoptosis in mud crab (Scylla paramamosain)

Potassium channel modulatory factor 1 (KCMF1), an E3 ubiquitin ligase, plays a vital role in renal tubulogenesis, preeclampsia, and tumor development in mammals. Nevertheless, the function of KCMF1 in invertebrates remains to be investigated. Here, we identified KCMF1-like from Scylla paramamosian, encoding 242 amino acids with two zinc finger domains at the N-terminal. Real-time quantitative PCR analysis revealed that KCMF1-like was expressed in all tested tissues, including hemocytes, brain, mid-intestine, subcuticular epidermis, gills, muscle, heart, and stomach, with higher levels in muscle and mid-intestine. KCMF1-like was up-regulated in the hemocytes of mud crabs challenged with white spot syndrome virus (WSSV). RNA interference (RNAi) was performed to investigate the impact of KCMF1-like on the proliferation of WSSV in mud crabs. Knock-down of KCMF1-like resulted in an increase of the WSSV copy number and an impairment of the hemocytes apoptosis rate in vivo. In addition, KCMF1-like could also affect the mitochondrial membrane potential. Collectively, these results revealed that KCMF1-like might play a crucial role in the defense against virus infection in mud crab. This study contributes a novel insight into the role of KCMF1-like in the antiviral immune defense mechanism in crustaceans.

WSSV exploits AMPK to activate mTORC2 signaling for proliferation by enhancing aerobic glycolysis

AMPK plays significant roles in the modulation of metabolic reprogramming and viral infection. However, the detailed mechanism by which AMPK affects viral infection is unclear. The present study aims to determine how AMPK influences white spot syndrome virus (WSSV) infection in shrimp (Marsupenaeus japonicus). Here, we find that AMPK expression and phosphorylation are significantly upregulated in WSSV-infected shrimp. WSSV replication decreases remarkably after knockdown of Ampkα and the shrimp survival rate of AMPK-inhibitor injection shrimp increases significantly, suggesting that AMPK is beneficial for WSSV proliferation. Mechanistically, WSSV infection increases intracellular Ca2+ level, and activates CaMKK, which result in AMPK phosphorylation and partial nuclear translocation. AMPK directly activates mTORC2-AKT signaling pathway to phosphorylate key enzymes of glycolysis in the cytosol and promotes expression of Hif1α to mediate transcription of key glycolytic enzyme genes, both of which lead to increased glycolysis to provide energy for WSSV proliferation. Our findings reveal a novel mechanism by which WSSV exploits the host CaMKK-AMPK-mTORC2 pathway for its proliferation, and suggest that AMPK might be a target for WSSV control in shrimp aquaculture.

Identification of a Double-β-Defensin with Multiple Antimicrobial Activities in a Marine Invertebrate.

β-Defensins are a family of cysteine-rich antimicrobial peptides that are generally monodomain. Interestingly, the avian β-defensin 11 (AvBD11) is unique, with two β-defensin motifs with a broad range of antimicrobial activities. However, a double-sized β-defensin has not been identified and functionally characterized in invertebrates. In this study, we cloned and identified a double-β-defensin in shrimp Litopenaeus vannamei (named LvDBD) and explored its potential roles during infection with shrimp pathogens Vibrio parahaemolyticus and white spot syndrome virus (WSSV). LvDBD is an atypical double-sized defensin, which is predicted to possess two motifs related to β-defensin and six disulfide bridges. The RNA interference-mediated knockdown of LvDBD invivo results in phenotypes with increased bacterial loads, rendering the shrimp more susceptible to V. parahaemolyticus infection, which could be rescued by the injection of recombinant LvDBD protein. In vitro, rLvDBD could destroy bacterial membranes and enhance hemocyte phagocytosis, possibly attributable to its affinity to the bacterial wall components LPS and peptidoglycan. In addition, LvDBD could interact with several viral envelope proteins to inhibit WSSV proliferation. Finally, the NF-κB transcription factors (Dorsal and Relish) participated in the regulation of LvDBD expression. Taken together, these results extend the functional understanding of a double-β-defensin to an invertebrate and suggest that LvDBD may be an alternative agent for the prevention and treatment of diseases caused by V. parahaemolyticus and WSSV in shrimp.

Isoferulic acid affords the antiviral potential and restrains white spot syndrome virus proliferation in crayfish (Procambarus clarkii)

White spot syndrome virus (WSSV), the causative agent of White spot disease (WSD), not only caused huge economic losses, but also greatly limited the development of crustacean breeding industry. Chinese herbs are widely used and have great application potential in antiviral research. In thist study, the antiviral effects of 11 Chinese herb extracts were evaluated in Procambarus clarkii (crayfish) model of WSSV infection, among which Cimicifuga foetida L. extract showed the highest inhibition rate (88.22%) against WSSV replication. On this basis, the anti-WSSV effect of Isoferulic acid (IFA), a major bioactive component of Cimicifuga foetida L., was further investigated. The results showed that IFA significantly inhibited WSSV replication (96.57%, 50 mg/kg) in a dose-dependent manner and significantly improved the survival of WSSV-challenged crayfish. IFA had the potential preventive and therapeutic effect against WSSV infection, and the preventive effect was better than the therapeutic effect. Mechanistically, IFA suppressed the expression of the immediate early gene ie1 , the DNA polymerase gene DNApol and envelope protein gene Vp28 to inhibit WSSV replication, activated TLR , CTL and ProPo genes expression to block ie1 transcription, and suppressed BAF , STAT , NF-κb and Crustin 1 genes expression to elevate innate immunity against WSSV infection. Besides, IFA upregulated the cMnSOD , mMnSOD , CAT and GST genes expression, and downregulated the COX-1 and COX-2 genes expression to balance the oxidative stress and inflammatory damage caused by WSSV infection. This study provides a theoretical basis and meaningful reference for the development of IFA as a highly effective and practical anti-WSSV drug.

First Discovery of Beta-Sitosterol as a Novel Antiviral Agent against White Spot Syndrome Virus.

The outbreak of white spot syndrome (WSS) is a looming challenge, due to dramatic losses to the crustacean aquaculture industry. However, at present, there are no prophylactic or therapeutic means to control this infectious viral disease. Here, we screened fifteen medicinal plants for their inhibitory activity on the white spot syndrome virus (WSSV), using red swamp crayfish (Procambarus clarkii) as a model species. The results showed that the crude extracts of Pinellia ternata (Thunb.) Breit. had the highest inhibitory effect (91.59%, 100 mg/kg) on WSSV proliferation, and its main component, beta-sitosterol, showed a much higher activity (95.79%, 50 mg/kg). Further, beta-sitosterol potently reduced (p < 0.01) viral loads and viral gene transcription levels in a concentration-dependent fashion, and significantly promoted the survival rate of WSSV-challenged crayfish (57.14%, 50 mg/kg). The co-incubation assay indicated that beta-sitosterol did not influence the infectivity of WSSV particles. Both pre- and post-treatment of beta-sitosterol exerted a significant inhibitory effect (p < 0.01) on the viral load in vivo. Mechanistically, beta-sitosterol not only interfered with the expression of viral genes (immediate early gene 1, ie1; DNA polymerase, DNApol) that are important in initiating WSSV transcription, but it also attenuated the hijacking of innate immune signaling pathways (Toll, IMD, and JAK/STAT pathways) by viral genes to block WSSV replication. Moreover, the expression of several antiviral immune, antioxidant, pro-inflammatory, and apoptosis-related genes changed significantly in beta-sitosterol-treated crayfish. Beta-sitosterol is a potent WSSV inhibitor and has the potential to be developed as an effective anti-WSSV agent against a WSS outbreak in crustacean aquaculture.

White spot syndrome virus directly activates mTORC1 signaling to facilitate its replication via polymeric immunoglobulin receptor-mediated infection in shrimp.

Previous studies have shown that the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway has antiviral functions or is beneficial for viral replication, however, the detail mechanisms by which mTORC1 enhances viral infection remain unclear. Here, we found that proliferation of white spot syndrome virus (WSSV) was decreased after knockdown of mTor (mechanistic target of rapamycin) or injection inhibitor of mTORC1, rapamycin, in Marsupenaeus japonicus, which suggests that mTORC1 is utilized by WSSV for its replication in shrimp. Mechanistically, WSSV infects shrimp by binding to its receptor, polymeric immunoglobulin receptor (pIgR), and induces the interaction of its intracellular domain with Calmodulin. Calmodulin then promotes the activation of protein kinase B (AKT) by interaction with the pleckstrin homology (PH) domain of AKT. Activated AKT phosphorylates mTOR and results in the activation of the mTORC1 signaling pathway to promote its downstream effectors, ribosomal protein S6 kinase (S6Ks), for viral protein translation. Moreover, mTORC1 also phosphorylates eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), which will result in the separation of 4EBP1 from eukaryotic translation initiation factor 4E (eIF4E) for the translation of viral proteins in shrimp. Our data revealed a novel pathway for WSSV proliferation in shrimp and indicated that mTORC1 may represent a potential clinical target for WSSV control in shrimp aquaculture.

Molecular characterization and expression of the autophagy-related gene Atg14 in WSSV-infected Procambarus clarkii

Atg14 (autophagy-related gene 14), also known as Atg14L or Barkor (Beclin-1 associated autophagy-related key regulator), plays an important role in a variety of biological processes including immunity, development, tumor inhibition, longevity, and protection against some cardiac and neurodegenerative diseases. However, very few studies have characterized Atg14 expression in invertebrates, particularly crustaceans. Here, a novel Atg14 gene from Procambarus clarkii (named PcAtg14) was characterized via RACE technology. Bioinformatics analysis showed that the total length of the PcAtg14 gene sequence was 2,880 bp, and it was predicted to encode 488 amino acids. The results of homology comparison showed that PcAtg14 exhibited the highest homology with crustacean the American lobster (Homarus americanus). Quantitative real-time PCR expression analysis showed that PcAtg14 was expressed in all tissues of P. clarkii, with the hepatopancreas having the highest expression and the eyestalk exhibiting the lowest expression. Upon white spot syndrome virus (WSSV) infection, the relative expression of PcAtg14 in the hepatopancreas, muscle, hemocyte, gill, heart and epidermis were significantly up-regulated at different time periods. After PcAtg14 gene silencing via RNA interference (RNAi), the proliferation of WSSV in P. clarkii was significantly inhibited, which coincided with a significant increase in P. clarkii mortality and an increase in the expression of autophagy-related genes (ATGs). Transmission electron microscopy analysis demonstrated an increase in the number of autophagosomes in the hepatopancreas of the PcAtg14 gene silencing group compared to the control group after WSSV infection. Collectively, these results indicated that PcAtg14 suppressed autophagy by reduce the fusion of autophagosomes and lysosomes, thereby promoting WSSV replication in P. clarkii. The findings here therefore provide novel insights into the immune mechanisms through which P. clarkii responds to WSSV infection.

Luteolin in Lonicera japonica inhibits the proliferation of white spot syndrome virus in the crayfish Procambarus clarkii

White spot disease caused by the white spot syndrome virus (WSSV) is a highly lethal infectious disease of crustacean aquaculture, with no effective means of control. Active compounds from herbal medicines are promising candidates for the development of antiviral agents for WSSV. We evaluated the efficacy of 12 herbal medicines in inhibiting WSSV proliferation in a WSSV infection model (Procambarus clarkii), Lonicera japonica extract had the strongest inhibitory effect on WSSV replication. Luteolin (LUT), the major bioactive compound of L. japonica extract, was further investigated and revealed to have a strong inhibitory effect on WSSV (96.04%, 50 mg/kg). LUT treatment decreased the WSSV load and the expression of important viral genes (immediate-early gene ie1, DNA polymerase DNApol and envelope protein Vp28) in a dose-dependent manner and increased the survival of WSSV-infected crayfish by 60.00% (50 mg/kg). Co-incubation and time-of-addition assays revealed that LUT did not affect the infectivity of viral particles or viral absorption and entry. Expression of immune-related genes such as barrier-to-autointegration (BAF), heat shock protein 70 (Hsp70) and signal transducer and activator of transcription (STAT), which may be beneficial for viral replication, were significantly decreased (P < 0.01) in LUT-treated crayfish. LUT also modified the expression of antioxidants (SOD, CAT and GST), pro-inflammatory factor (COX-1 and COX-2), and apoptosis-related (BAX and BI-1) genes (P < 0.01) to enhance antioxidant defenses, mitigate inflammation and induce apoptosis, respectively. Overall, LUT could be used as a prophylactic or therapeutic agent against WSSV infection in crustacean aquaculture.

A IFI27 gene contributes to ER-stress mediated apoptosis and benefits for white spot syndrome virus infection in Litopenaeus vannamei

The interplay between virus and host has been one of the hot spot in virology, and it is also the important aspect of revealing the mechanism of virus infection. Increasing studies revealed that several key molecules took part in the process of virus-host interaction. White spot syndrome virus (WSSV) has been proved to affect several physiological processes of the host cells, especially apoptosis. While the relationship between them still remains unclear. In this study, a IFI27 gene (LvIFI27) of Litopenaeus vannamei was cloned. It is indicated that LvIFI27 was induced upon endoplasmic reticulum (ER)-stress and unfolded protein response activator Thapsigargin. Unlike human IFI27 locating to mitochondria, LvIFI27 lied to ER, and was involved in cell apoptosis process. Moreover, results of cumulative mortality analysis showed that LvIFI27 might contributed to WSSV proliferation by promoting apoptosis during the process of viral infection. Findings in this study enriched our understanding of the relationship between WSSV infection and ER-stress mediated apoptosis.

Acyclovir inhibits white spot syndrome virus replication in crayfish Procambarus clarkii

White spot syndrome virus (WSSV) is a fatal pathogen threatening global crustacean industry with no commercially available drugs to control WSSV. To address the urgent need for finding effective antiviral agents against WSSV, we examined the anti-WSSV activities of 11 common antiviral agents in crayfish Procambarus clarkia. The results showed that acyclovir displayed the highest inhibition on WSSV replication in vivo (92.59%, 50 mg/kg). Acyclovir repressed WSSV proliferation followed a dose-dependent fashion and pre- or post-treatment of acyclovir exerted strong inhibition on the viral loads. Further, we observed a markedly reduced expression levels of WSSV genes (immediate-early IE gene ie1, DNA polymerase gene DNApol and envelope protein gene Vp28) that are crucial in viral life cycle with the acyclovir treatment during the early infection. Meantime, we also found a significantly increased expressions of anti-oxidative as well as apoptosis related genes, suggesting that acyclovir could effectively suppress WSSV replication in vivo. Finally, acyclovir treatment could significantly improve the survival rate of WSSV-challenged crayfish by 56%. Taken together, acyclovir has the potential to be developed as a promising preventive or therapeutic agent against WSSV infection, and this finding may provide a reference for rapid discovery anti-WSSV agent in crustacean aquaculture.

Evaluation of the antiviral activity of naringenin, a major constituent of Typha angustifolia, against white spot syndrome virus in crayfish Procambarus clarkii.

White spot syndrome virus (WSSV) is a serious pathogen threatening global crustacean aquaculture with no commercially available drugs. Herbal medicines widely used in antiviral research offer a rich reserve for drug discovery. Here, we investigated the inhibitory activity of 13 herbal medicines against WSSV in crayfish Procambarus clarkii and discovered that naringenin (NAR) has potent anti-WSSV activity. In the preliminary screening, the extracts of Typha angustifolia displayed the highest inhibitory activity on WSSV replication (84.62%, 100mg/kg). Further, NAR, the main active compound of T.angustifolia, showed a much higher inhibition rate (92.85%, 50mg/kg). NAR repressed WSSV proliferation followed a dose-dependent manner and significantly improved the survival of WSSV-challenged crayfish. Moreover, pre- or post-treatment of NAR displayed a comparable inhibition on the viral loads. NAR decreased the transcriptional levels of vital genes in viral life cycle, particularly for the immediately early-stage gene ie1. Further results showed that NAR could decrease the STAT gene expression to block ie1 transcription. Besides, NAR modulated immune-related gene Hsp70, antioxidant (cMnSOD, mMnSOD, CAT, GST), anti-inflammatory (COX-1, COX-2) and pro-apoptosis-related factors (Bax and BI-1) to inhibit WSSV replication. Overall, these results suggest that NAR may have the potential to be developed as preventive or therapeutic agent against WSSV.

Herbicide atrazine exposure induce oxidative stress, immune dysfunction and WSSV proliferation in red swamp crayfish Procambarus clarkii

Atrazine is considered as a potential environmental endocrine disruptors and exhibits various toxic effects on animals. It has a great impact in the aquatic ecosystems, but there are few studies on its immunotoxicity in crustaceans. In the present study, the Procambarus clarkii were utilized to assess the immune toxicity after 0.5 mg/L and 5 mg/L atrazine exposure. A significant decrease in total hemocytes count (THC) was observed at 5 mg/L atrazine exposure throughout the experiment. The activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were significantly inhibited, but the content of reactive oxygen species (ROS) and malondialdehyde (MDA) were up-regulated, indicating the potential oxidative stress. The analysis of the integrated biomarker response (IBR) showed the induction of oxidative stress biomarkers and the inhibition of antioxidants. After 5 mg/L atrazine exposure for 144 h, the integrity of crayfish hepatopancreas was destroyed with disappeared connections between tubules and increased liver tubules vacuoles. The relative expression levels of different immune genes in hepatopancreas after atrazine exposure were measured. Most of these genes were suppressed and exhibited a certain dose-dependent effect. The results of crayfish white spot syndrome virus (WSSV) replication shown the amount of virus in muscle was significantly higher and exhibited a higher mortality rate at 5 mg/L group than other groups. The present study determined the impact of atrazine exposure on WSSV outbreaks, and also provide an important basis for further assessing the occurrence of pesticides on diseases of P. clarkii.

Identification of a Shrimp E3 Ubiquitin Ligase TRIM50-Like Involved in Restricting White Spot Syndrome Virus Proliferation by Its Mediated Autophagy and Ubiquitination.

Most tripartite motif (TRIM) family proteins are critical components of the autophagy machinery and play important roles in host defense against viral pathogens in mammals. However, the roles of TRIM proteins in autophagy and viral infection have not been studied in lower invertebrates, especially crustaceans. In this study, we first identified a TRIM50-like gene from Penaeus monodon (designated PmTRIM50-like), which, after a white spot syndrome virus (WSSV) challenge, was significantly upregulated at the mRNA and protein levels in the intestine and hemocytes. Knockdown of PmTRIM50-like led to an increase in the WSSV quantity in shrimp, while its overexpression led to a decrease compared with the controls. Autophagy can be induced by WSSV or rapamycin challenge and has been shown to play a positive role in restricting WSSV replication in P. monodon. The mRNA and protein expression levels of PmTRIM50-like significantly increased with the enhancement of rapamycin-induced autophagy. The autophagy activity induced by WSSV or rapamycin challenge could be inhibited by silencing PmTRIM50-like in shrimp. Further studies showed that rapamycin failed to induce autophagy or inhibit WSSV replication after knockdown of PmTRIM50-like. Moreover, pull-down and in vitro ubiquitination assays demonstrated that PmTRIM50-like could interact with WSSV envelope proteins and target them for ubiquitination in vitro. Collectively, this study demonstrated that PmTRIM50-like is required for autophagy and is involved in restricting the proliferation of WSSV through its ubiquitination. This is the first study to report the role of a TRIM family protein in virus infection and host autophagy in crustaceans.

Immune toxicity of phenanthrene and its combined effects of white spot syndrome virus on the survival of kuruma shrimp (Penaeus Japonicus)

Shrimp inhabiting coasts that are frequented by humans are exposed to various pollutants. Additionally, viral infections that cause serious damage to shrimp populations have been observed in these environments. The present study sought to evaluate the immunotoxic effects of phenanthrene (Phe), a pollutant detected in coastal environments, on kuruma shrimp (Penaeus japonicus). We further examined the survival of shrimp following combined exposure to Phe (30 or 300 μg/L) and white spot syndrome virus (WSSV). Results show that exposure to Phe for seven days decreased immune system-related parameters, including total hemocyte count and phenoloxidase activity in hemolymph (p < 0.05). However, these effects were not detected after three days of exposure. Moreover, a combined exposure assay revealed that shrimp mortality increased following exposure to 300 μg/L Phe and infection with WSSV. The number of WSSV gene copies was also observed to increase in these co-exposed shrimp. Taken together, these results indicate that long-term Phe exposure impairs the immune system of P. japonicus, resulting in fatal proliferation of WSSV. Hence, considering that combined exposure to Phe and WSSV leads to increased mortality of shrimp, it is imperative that the detrimental effects elicited by multiple stresses be considered, and controlled, in areas inhabited by kuruma 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.

Sp-CBL inhibits white spot syndrome virus replication by enhancing apoptosis in mud crab (Scylla paramamosain)

In mammals, casitas B-lineage lymphoma (CBL) family proteins, a RING-type E3 ubiquitin ligase, are involved in many signal transduction pathways. However, the functions of CBL in invertebrates are not well elucidated. In this study, Sp-CBL containing CBL-N, CBL-2, CBL-3 and RING domains was identified in mud crab Scylla paramamosain. Sp-CBL was widely expressed in all tissues tested and found to be significantly up-regulated in the hemocytes of mud crab challenged by white spot syndrome virus (WSSV). The RNA interference of Sp-CBL increased the copy number of WSSV and declined the apoptosis rate of hemocytes. In addition, Sp-CBL could affect the activities of caspase 3 and the mitochondrial membrane potential. Taken together, the results of this study revealed that Sp-CBL could restrict WSSV proliferation through enhancing the apoptosis of the hemocytes, which would provide a novel insight into the anti-viral response in the innate immunity system of mud crab.

Survival and immune response of white shrimp Litopenaeus vannamei following single and concurrent infections with WSSV and Vibrio parahaemolyticus

The survival and immune responses of Litopenaeus vannamei were evaluated during white spot syndrome virus (WSSV) or Vibrio parahaemolyticus single and concurrent infections. The mortality, WSSV load, activities of 4 immune enzymes: acid phosphatase (ACP), alkaline phosphatase (AKP), peroxidase (POD) and superoxide dismutase (SOD), and the transcription of Evolutionarily Conserved Signaling Intermediate in Toll pathways of L.vannamei (LvECSIT) were quantified at 0, 3, 6, 12, 24, 48, 72 and 96 h post-infection (pi). The results showed: (i) the cumulative mortality of the co-infection group (WSSV and V. Parahaemolyticus 83%) was significantly lower than the WSSV infection group (97%) (P < 0.05) at 96 hpi; (ii) copies of WSSV in the co-infection group were significantly lower than that of the single infection group from 24 to 96 hpi (P < 0.05); (iii) ACP, AKP,POD and SOD activity in the gills of the co-infection group was higher than that of the WSSV group at12, 48 and 96 hpi (P < 0.05).The expression of LvECSIT mRNA in the co-infection group was significantly higher than in the WSSV infection group from 12 to 72 hpi (P < 0.05).The results indicate that proliferation of WSSV is inhibited by V.parahaemolyticus infection. In addition, infection with WSSV alone causes a significant reduction in some immune responses of shrimp than co-infection with WSSV and V.parahaemolyticus occurs at 26 °C. Third, LvECSIT, an essential member of TLR signaling pathway might play a crucial role in shrimp defense against WSSV – Vibrio co-infection.

Proliferation dynamics of WSSV in crayfish, Procambarus clarkii, and the host responses at different temperatures.

The replication profile of white spot syndrome virus (WSSV) in crayfish, Procambarus clarkii, at different water temperature was investigated in this study. The WSSV detections were negative at 15±1°C, and the natural infection ratio increased at 19±1°C (24.2%±2.25%), reached 100% at 25±1°C and decreased at 30±1°C (93.2%±3.37%). The WSSV genome copies number was much higher at 25±1°C (≥5×106.45±0.35 /mg) than at 15±1°C (≤5×101.13±0.12 /mg), 19±1°C (≤5×102.74±0.48 /mg) and 32±1°C (≤5×103.18±0.27 /mg). Meanwhile, the significant transcription signals of immediate early gene ie1 and late gene vp28 and a large number of virus particles were detected in epitheliums of stomach, gut and gill, hepatopancreas, heart and muscle cells at 25±1°C by using in situ hybridization (ISH) and transmission electron microscopy. The experimental infection of P.clarkii with WSSV infection showed reduced mortality and lower virus copies number at 19±1°C (23.51%±0.84%, ≤5×103.41±0.11 /mg) and 32±1°C (38.42%± 1.21%, ≤5×103.72±0.13 /mg) compared to 25±1°C (100%, ≥5×104.99±0.24 /mg). The water temperature regulated the transcription of immune-related genes (crustin2, prophenoloxidase (proPO) and heat shock protein70 (Hsp70)), with some differences between WSSV treatments and control treatments. These results demonstrate that water temperature has effect on WSSV proliferation, which may due to transcriptional response of immune-related genes to temperature.

Scavenger receptor B promotes bacteria clearance by enhancing phagocytosis and attenuates white spot syndrome virus proliferation in Scylla paramamosian

Phagocytosis and apoptosis are key cellular innate immune responses against bacteria and virus in invertebrates. Class B scavenger receptors (SRBs), which contain a CD36 domain, are critical pattern recognition receptors (PRRs) of phagocytosis for bacteria and apoptotic cells. In the present study, we identified a member of SRB subfamily in mud crab Scylla paramamosain, named Sp-SRB. The full-length cDNA of Sp-SRB is 2593 bp with a 1629 bp open reading frame (ORF) encoding a putative protein of 542 amino acids, and predicted to contain a CD36 domain with two transmembrane regions at the C- and N-terminals. Real-time qPCR analysis revealed that Sp-SRB was widely expressed in all tissues tested, and the expression of Sp-SRB was up-regulated upon challenge with Vibrio parahaemolyticus, white spot syndrome virus (WSSV), lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (PolyI:C). Moreover, in vitro experiments indicated that recombinant Sp-SRB protein (rSp-SRB) could bind to fungi, Gram-positive and Gram-negative bacteria. RNA interference of Sp-SRB resulted in significant reduction in the expression level of phagocytosis related genes, antimicrobial peptides (AMPs) and Toll-like receptors (TLRs), which consequently led to impairment in both bacterial clearance and the phagocytotic activity of hemocytes. In addition, we found that Sp-SRB had the ability to attenuate the replication of WSSV proliferation in mud crab S. paramamosain. Collectively, this study has shown that Sp-SRB contributed to bacteria clearance by enhancing phagocytosis and up-regulating the expression of AMPs possibly in a TRLs (SpToll 1 and SpToll 2)-dependent manner. Besides, Sp-SRB inhibited the replication of WSSV in S. paramamosian probably through enhancement of hemocytes phagocytosis of apoptotic cells.