White Spot Syndrome Virus Replication Research Articles

A novel nitrogenous coumarin: The promising antiviral therapy against white spot syndrome virus in the culture of shrimp seedlings

White spot disease (WSD), attributed to the white spot syndrome virus (WSSV), represents a substantial danger to the international shrimp sector, resulting in considerable losses in both production and economic terms. Nevertheless, there are currently no approved therapeutic measures to control the outbreak. This study involved the synthesis of 15 new compounds for the assessment of their anti-WSSV efficacy in Litopenaeus vannamei post-larvae. Among these compounds, N-(4-methyl-2-oxo-2H-chromen-7-yl)acetamide (P13) exhibited a maximum antiviral response of > 90 %, with a median effective concentration (EC50) < 12 mg/L, making P13 the most promising antiviral compound. In the cases where the larvae were simultaneously incubated with WSSV and P13, as well as where the larvae were first infected with WSSV and then treated with P13, the replication of WSSV was significantly inhibited. However, pre-treatment with P13 had minimal impact on WSSV multiplication, suggesting a potential therapeutic effect. Simultaneously, the cumulative mortality rate of WSSV-infected post-larvae decreased in the presence of P13. After the shrimp larvae were infected with WSSV, continuously changing the aquacultural water containing DMSO resulted in a mortality rate of 80 % at 120 hours post-infection (hpi); whereas continuously changing the aquaculture water containing P13 resulted in a mortality rate of 45 %. Given that P13 remains stable in farmed water for up to two days, it is recommended to regularly replace the medication to eliminate the virus threat. In conclusion, P13 demonstrates a positive anti-WSSV activity and holds potential for further development as an antiviral therapy against WSSV infection.

The antimicrobial peptide Sparamosin26–54 exhibits antiviral activity against three aquatic enveloped viruses through lipid-binding-mediated virus lysis

The aquaculture industry is faced with huge economic losses caused by various enveloped virus infections, such as white spot syndrome virus (WSSV), Rana grylio virus (RGV), and Anguillid Herpesvirus 1 (AngHV). The urgent need for readily available broad-spectrum antiviral agents has led to the exploration of antiviral peptides (AVPs), among which antimicrobial peptides (AMPs) with antiviral activity have attracted increasing attention. Sparamosin26–54 (derived from Scylla paramamosain) is a promising antimicrobial agent. In this study, we found that Sparamosin26–54 significantly inhibited WSSV transcription and replication (as indicated by VP28 gene and protein levels), irreversibly inactivated virions, and impeded viral entry before binding to the cell. Transmission electron microscopy observations showed that Sparamosin26–54 had a direct destructive effect on WSSV virions. Lipidomic analysis and ELISA assay highlighted that Sparamosin26–54 had a potent affinity for phosphatidic acid (PA) and phosphatidylserine (PS) of WSSV. Liposome leakage assays further revealed a pronounced effect of Sparamosin26–54 on membrane lipid compositions, emphasizing its efficacy against the other two enveloped viruses (RGV and AngHV), but not against the non-enveloped virus, American eel adomavirus (AEAdov). Notably, in three animal models of viral infection, Sparamosin26–54 reduced viral loads in tissues, increased survival, or decreased infection rates in a concentration-dependent manner. Importantly, Sparamosin26–54 showed no animal toxicity under different modes of administration and could maintain antiviral activity in the complex aquatic environment. Taken together, Sparamosin26–54 was proved to be effective against three enveloped viruses, possibly inducing viral lysis via lipid-binding, providing an alternative approach for combating enveloped virus infections.

WSSV early protein WSSV004 enhances viral replication by suppressing LDH activity

White spot syndrome virus (WSSV) is known to upregulate glycolysis to supply biomolecules and energy for the virus's replication. At the viral genome replication stage, lactate dehydrogenase (LDH), a glycolytic enzyme, shows increased activity without any increase in expression. In the present study, yeast 2-hybrid screening was used to identify WSSV proteins that interacted with LvLDH isoform 1 and 2, and these included the WSSV early protein WSSV004. The interaction between WSSV004 and LvLDH1/2 was confirmed by co-immunoprecipitation. Immunofluorescence showed that WSSV004 co-localized with LvLDH1/2 in the cytoplasm. dsRNA silencing experiments showed that WSSV004 was crucial for WSSV replication. However, although WSSV004 silencing led to the suppression of total LvLDH gene expression during the viral late stage, there was nevertheless a significant increase in LvLDH activity at this time. We also used affinity purification-mass spectrometry to identify cellular proteins that interact with WSSV004, and found a total of 108 host proteins and 3 WSSV proteins with which it potentially interacts. Bioinformatics analysis revealed that WSSV004 and its interacting proteins might be responsible for various biological pathways during infection, including vesicular transport machinery and RNA-related functions. Collectively, our study suggests that WSSV004 serves as a multifunctional modulator to facilitate WSSV replication.

Molecular Characterization and Expression Analysis of the C-Type Lectin Domain Family 4 Member F in Litopenaeus vannamei against White Spot Syndrome Virus.

White spot disease (WSD) outbreaks pose a significant threat to the Pacific white shrimp (Litopenaeus vannamei) farming industry. The causative agent is the white spot syndrome virus (WSSV). There are no effective treatments for WSD so far. Therefore, understanding the resistance mechanisms of L. vannamei against the WSSV is crucial. C-type lectins (CTLs) are important pattern recognition receptors (PRRs) that promote agglutination, phagocytosis, encapsulation, bacteriostasis, and antiviral infections. This study cloned the C-type lectin domain family 4 member F (LvCLEC4F) from L. vannamei. LvCLEC4F contains a 492 bp open reading frame (ORF) encoding a protein of 163 amino acids, including a carbohydrate recognition domain (CRD). Following a challenge with the WSSV, the expression profile of LvCLEC4F was significantly altered. Using RNA interference (RNAi) technology, it was found that LvCLEC4F promotes WSSV replication and affects the expression levels of genes related to the regulation of apoptosis, signaling and cellular stress response, and immune defense. Meanwhile, the hemolymph agglutination phenomenon in vivo was weakened when LvCLEC4F was knocked down. These results indicated that LvCLEC4F may play an important role in the interaction between L. vannamei and WSSV.

PHB2 inhibits WSSV replication by promoting the nuclear translocation of STAT

Prohibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses and cell proliferation. However, the function of the PHBs in immune regulation has largely not been determined. In the present study, we identified PHB2 in the red swamp crayfish Procambarus clarkii. PHB2 was found to be widely distributed in several tissues, and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge. PHB2 significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. Here, we observed that PHB2 promotes the nuclear translocation of STAT by binding to STAT. After blocking PHB2 or STAT with antibodies or interfering with PHB2 or STAT, the expression levels of the antiviral genes β-thymosin (PcThy-4) and crustin2 (Cru2) decreased. The gene sequence of PHB2 was analyzed and found to contain a nuclear introgression sequence (NIS). After in vivo injection of PHB2 with deletion of NIS (rΔNIS-PHB2), the nuclear translocation of STAT did not change significantly compared to that in the control group. These results suggest that PHB2 promoted the nuclear translocation of STAT through NIS and mediated the expression of antiviral proteins to inhibit WSSV infection.

Potential application of a small molecule compound in aquaculture against WSSV infection by maintaining the homeostasis of intestinal microbiota

Disease outbreaks consistently lead to substantial economic losses in crustacean farming due to the absence of appropriate treatments for infections caused by the white spot syndrome virus (WSSV). This study focused on examining the in vivo antiviral effects of 2-amino-4-(4-chlorophenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3‑carbonitrile (C7) as well as its impact on the intestinal microbiota of shrimp infected with WSSV. The findings demonstrated that C7 at a dosage of 50 mg/kg markedly suppressed WSSV replication in adult shrimp, achieving a maximum inhibitory rate exceeding 90%. Furthermore, C7 effectively inhibited the horizontal transmission of WSSV. From a histopathological perspective, C7 demonstrated the ability to mitigate the damage caused by WSSV to intestinal tissues, preserving the integrity of both the intestinal wall and villi. Infections with WSSV led to a reduction in the abundance of Firmicutes and an elevation in Proteobacteria levels. At the genus level, certain beneficial bacterial groups, including Muribaculaceae and Colidextribacter, exhibited diminished abundance, whereas the presence of opportunistic bacteria such as Shiwanella and Vibrio increased in response to WSSV infection. Following treatment with C7, the abundance of beneficial bacteria increased, harmful bacteria decreased, and the structure of the bacterial community was restored to a normalized state. PICRUSt analysis revealed that the modified microbiota due to WSSV primarily pertained to cellular processes and metabolism, implying heightened activity of gut microbes in afflicted shrimp. In contrast, C7 exhibited the capacity to restore the functionality of the gut microbiota in diseased shrimp. In summary, C7 potentially combats WSSV infection by preserving intestinal tract morphology and upholding a typical flora structure. This groundwork supports the potential application of C7 as a specialized antiviral treatment in aquaculture.

Lectin diversity and their positive roles in WSSV replication through regulation of calreticulin expression and inhibiting ALFs expression

In biological evolution, gene duplication (GD) generates new genes to facilitate new functions. C-type lectins (CTLs) in crayfish have been extended by GD to expand their family members. In this study, four CTL genes generated by GD were identified from Procambarus clarkii (PcLec1–4). Among these four genes, PcLec1 can also generate new isoforms with different numbers of tandem repeats through DNA slip mispairing. PcLec1–4 was widely expressed in multiple tissues. The expression levels of PcLec1–4 were upregulated in the intestine of P. clarkii upon white spot syndrome virus (WSSV) challenge at multiple time points. Further analysis indicated that GATA transcription factor regulated PcLec1–4 expression. RNA interference and recombinant PcLec1–4 protein injection experiments suggested that PcLec1–4 promoted the expression of calreticulin (PcCRT) and negatively regulated the expression of antimicrobial peptides, thereby promoting WSSV replication. This study contributes to the understanding of the function of CTLs produced by GD during WSSV invasion in crustaceans.

Shrimp SIRT4 promotes white spot syndrome virus replication

In WSSV pathogenesis, the molecular mechanisms and the key host factors that regulate the viral replication and morphogenesis remain unclear. However, like most viruses, WSSV is known to induce metabolic reprogramming in several metabolic pathways including the host glutamine metabolism, and several recent reports have suggested that the sirtuins SIRT3, SIRT4, and SIRT5, which belong to a family of NAD+-dependent deacetylases, play an important role in this regulation. Here we focus on characterizing LvSIRT4 from Litopenaeus vannamei and investigate its role in regulating glutamine dehydrogenase (GDH), an important enzyme that promotes glutaminolysis and viral replication. We found that LvSIRT4 silencing led to significant decreases in both WSSV gene expression and the number of viral genome copies. Conversely, overexpression of LvSIRT4 led to significant increases in the expression of WSSV genes and the WSSV genome copy number. Immunostaining in Sf9 insect cells confirmed the presence of LvSIRT4 in the mitochondria and the co-localization of LvSIRT4 and LvGDH in the same cellular locations. In vivo gene silencing of LvSIRT4 significantly reduced the gene expression of LvGDH whereas LvSIRT4 overexpression had no effect. However, neither silencing nor overexpression had any effect on the protein expression levels of LvGDH. Lastly, although GDH activity in uninfected shrimp was unchanged, the GDH enzyme activity in WSSV-infected shrimp was significantly increased after both LvSIRT4 silencing and overexpression. This suggests that although there may be no direct regulation, LvSIRT4 might still be able to indirectly regulate LvGDH via the mediation of one or more WSSV proteins that have yet to be identified.

White spot syndrome virus hijacks host PP2A-FOXO axes to promote its propagation

Viruses have developed superior strategies to escape host defenses or exploit host components and enable their infection. The forkhead box transcription factor O family proteins (FOXOs) are reportedly utilized by human cytomegalovirus during their reactivation in mammals, but if FOXOs are exploited by viruses during their infection remains unclear. In the present study, we found that the FOXO of kuruma shrimp (Marsupenaeus japonicus) was hijacked by white spot syndrome virus (WSSV) during infection. Mechanistically, the expression of leucine carboxyl methyl transferase 1 (LCMT1) was up-regulated during the early stages of WSSV infection, which activated the protein phosphatase 2A (PP2A) by methylation, leading to dephosphorylation of FOXO and translocation into the nucleus. The FOXO directly promoted transcription of the immediate early gene, wsv079 of WSSV, which functioned as a transcriptional activator to initiate the expression of viral early and late genes. Thus, WSSV utilized the host LCMT1-PP2A-FOXO axis to promote its replication during the early infection stage. We also found that, during the late stages of WSSV infection, the envelope protein of WSSV (VP26) promoted PP2A activity by directly binding to FOXO and the regulatory subunit of PP2A (B55), which further facilitated FOXO dephosphorylation and WSSV replication via the VP26-PP2A-FOXO axis in shrimp. Overall, this study reveals novel viral strategies by which WSSV hijacks host LCMT1-PP2A-FOXO or VP26-PP2A-FOXO axes to promote its propagation, and provides clinical targets for WSSV control in shrimp aquaculture.

White spot syndrome virus achieves early infection through Snail2 suppression of Relish nuclear translocation

In vertebrates, Snails are evolutionarily conserved and consist of 4–6 Cys2-His2 (C2H2)-Zinc finger domains. The key role of the Snails is well illustrated in cancer, but its role in immunity is limited. Two Snail2 genes, named Snail2α and Snail2β, containing 5 C2H2 at C-terminus, were identified in Procambarus clarkii. Snail2α was highly expressed in heart, gills and intestine, and Snail2β in gills, stomach and intestine. Decreased expression of Snail2α and Snail2β in gills and intestine at 6 h after White spot syndrome virus (WSSV) infection. Early WSSV replication levels are inhibited in dsSnail2α or dsSnail2β-injected crayfish compared to dsGFP-injected crayfish. The early WSSV replication levels are increased in rSnail2α or rSnail2β-injection crayfish. Further studies revealed that both Snail2α and Snail2β regulate the nuclear translocation of Relish. Relish entry into the nucleus promotes the expression of the WSSV immediate-early genes ie1 and ie15. WSSV abducts the host Snail2α and Snail2β in an unknown manner, facilitating the nuclear translocation of Relish and inducing ie1 and ie15 transcription. Crayfish responds to this escape mechanism of WSSV by reducing the expression of Snail2α and Snail2β at early stage of WSSV infection. This study provides new insights in immune response of crayfish to WSSV escape.

Antiviral, antioxidant, and anti-inflammatory activities of rhein against white spot syndrome virus infection in red swamp crayfish (Procambarus clarkii).

Infection caused by white spot syndrome virus (WSSV) leads to massive mortality and huge economic losses in the crustacean aquaculture industry; however, to date, no effective control means are available to control the disease. Thus, to better understand the pathological aspects of WSSV and to find effective compounds that have the potential to be developed as anti-WSSV agents are imperative. Herein, based on screening of plants with antiviral activity, we found that rhein, an anthraquinone compound in Rheum palmatum L., exhibited strong activity against WSSV infection in a red swamp crayfish (Procambarus clarkii) model. The results showed that rhein could dose dependently inhibit transcription of WSSV ie1, DNApol, and Vp28 genes, reduce viral load in vivo, and significantly improve the survival rate of WSSV-infected crayfish. Through investigation of the mechanisms of action, we found that rhein did not affect the infectivity of viral particles in vitro as well as the entry and release process in vivo. Alternatively, rhein could activate JAK/STAT and NF-κB pathways, significantly decrease the expression of crustin-1 and BAF but increase the expression of C-type lectin and proPO to arrest WSSV replication. Moreover, rhein also attenuated WSSV-induced oxidative and inflammatory stresses by regulating the expression of antioxidant and anti-inflammatory-related genes and enhanced innate immunity by reducing total protein levels while increasing the phosphatase activity. Taken together, our study shows rhein as a potent WSSV inhibitor, which provides great potential for the development of prophylaxis and therapy agents for controlling WSSV infection in crustacean aquaculture.IMPORTANCEAquaculture is essential for ensuring global food security by providing a significant source of animal protein. However, the spread of the white spot syndrome virus (WSSV) has resulted in considerable economic losses in crustacean industries. In this study, we evaluated the antiviral activity of rhein, the primary bioactive component of Rheum palmatum L., against WSSV infection, and many pathological aspects of WSSV were also described for the first time. Our mechanistic studies indicated that rhein effectively arrested the replication of WSSV in crayfish by modulating innate immunity to inhibit viral gene transcription. Furthermore, we observed that rhein attenuated WSSV-induced oxidative and inflammatory stresses by regulating the expression of antioxidant and anti-inflammatory-related genes while enhancing innate immunity by reducing total protein levels and increasing phosphatase activity. Our findings suggest that rhein holds great promise as a potent antiviral agent for the prevention and treatment of WSSV in aquaculture.

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.

Protection of Pacific white shrimp (Penaeus vannamei) against white spot syndrome virus infection by nitric oxide-generating compound S-nitrosoglutathione

Nitric oxide (NO), a highly reactive free radical generated endogenously by nitric oxide synthase (NOS), plays crucial roles in immune defense against a broad range of pathogens including viruses, bacteria, fungi, and protozoans. However, the regulation and effect of NO during viral infection in invertebrates remain largely unexplored. White spot syndrome virus (WSSV) is one of the most severe pathogens affecting the global shrimp farming industry. Our study shows that NO production in Pacific white shrimp Penaeus vannamei decreases during WSSV infection. Reducing NO production using the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) significantly promotes WSSV replication in primary culture of hemocytes isolated from P. vannamei. This effect is partically reversed by L-arginine, a native substrate for NOS, indicating that L-NAME's immunity-reducing action is NO-dependent. In vitro studies also reveal inhibition of WSSV replication in the presence of three NO donors: S-nitrosoglutathione (GSNO), S-nitro-N-acetylpenicillamine (SNAP), and diethylenetriamine nitric oxide adduct (DETA/NO). The involvement of NO in anti-WSSV protection is further demonstrated by an in vivo study where dietary supplementation with GSNO at non-toxic doses increases NO levels and reduces viral load and mortality in WSSV-challenged shrimp. Our results contribute to understanding the antiviral activity of NO in invertebrates and provide a theoretical basis for developing NO-based therapeutic strategies to control WSSV infection.

WSV403 suppresses JAK/STAT signaling to impair antiviral resistance in Pacific white shrimp Penaeus vannamei

Viral immediate early (IE) genes are key regulators of host antiviral defenses. White spot syndrome virus (WSSV) IE gene WSV403 encodes an E3 ligase which regulates the latency state of WSSV. However, the role of WSV403 in host immune response remains unclear. Here, we generated a transgenic Drosophila line that overexpresses WSV403 and found that WSV403 suppresses the JAK/STAT pathway and its downstream genes, leading to increased susceptibility and viral load upon Drosophila C virus infection. We further showed that WSV403 suppresses Stat92E expression without affecting its phosphorylation or degradation, suggesting a novel mechanism of JAK/STAT inhibition. Moreover, we demonstrated that knockdown of WSV403 in the Pacific white shrimp Penaeus vannamei reduces WSSV replication and enhances transcript levels of JAK/STAT pathway components. Our results revealed the immune modulatory function of WSV403 and uncovered a new mechanism by which WSSV inhibits host JAK/STAT signaling to impair antiviral resistance against WSSV infection.

Molecular characterization and functional analysis of TRIM37 from black tiger shrimp (Penaeus monodon).

The family of TRIM proteins with E3 ubiquitin ligase activity served important roles in the regulation of innate immune processes, in particular antiviral and proinflammatory cytokine responses. In this study, a novel TRIM37 homolog was identified from Penaeus monodon (named PmTRIM37). The PmTRIM37 protein contained three conserved domains (one RING finger domain, a B-box, and one Coiled-coil region) at its N-terminal and one Meprin and MATH domain at its C-terminal. The MATH domain was the characteristic of TRIM37 family. PmTRIM37 has relatively high expression in immune-related tissues such as hepatopancreas, gills, lymphoid organs and hemocytes. The expression levels of PmTRIM37 in hepatopancreas and lymphoid organs were significantly up-regulated after white spot syndrome virus (WSSV) infection. Knock down of PmTRIM37 promoted WSSV replication and VP28 expression, suggesting that PmTRIM37 played a negative role in WSSV infection. Further studies revealed that PmTRIM37 positively regulated the NF-κB pathway and Antimicrobial peptides (AMP) expression during WSSV infection. These findings indicated that PmTRIM37 might restrict WSSV replication by positively regulating NF-κB pathway during WSSV infection in P. monodon.

WSSV latency is maintained by a dynamic balance of host immune factors and disturbed by exposure to bacterial infection and low salinity

Shrimp is one of the primary aquaculture species worldwide. The white spot syndrome virus (WSSV) is a highly lethal, stress-dependent virus that has led to enormous economic losses in shrimp aquaculture. WSSV can trigger a persistent infection in shrimp (latency), which can be reactivated to become an acute infection following various stimuli. However, the molecular mechanisms underlying the WSSV latency establishment and the induction of reactivation are poorly understood. A WSSV latency model using low multiplicity infection via intramuscular injection was developed in this study. This research examined the effects of multiple host factors and stimuli on WSSV latency. These effects generated latency reactivation in this model. The outcomes revealed that low multiplicity infection via intramuscular injection could be applied to establish WSSV latency in shrimp. Besides, the expression levels of host genes IRF, Dorsal and Relish were found to be substantially correlated with viral loads during WSSV latent infection in vivo. The knockdown of IRF resulted in elevated viral loads and rendered the reactivation of WSSV latency to acute infection that caused mass mortality among shrimp. Furthermore, several bacterial infection and low salinity stress could induce WSSV transitions from latent to acute infection. Noteworthily, the expression levels of Dorsal and Relish, which are considered to promote the replication of WSSV, are upregulated following bacterial infection and low salinity stress. These results indicated that a delicate balance between host and WSSV interaction regulates the latency establishment. This balance could be broken by several stimuli, such as bacterial infection and low salinity stress, which will provide insights into developing management strategies for WSSV prevention and control.

The effect of white spot syndrome virus (WSSV) envelope protein VP28 on innate immunity and resistance to white spot syndrome virus in Cherax quadricarinatus

VP28 is the most abundant membrane protein of WSSV, and the recombinant protein VP28 (VP26 or VP24) was constructed for the immune protection experiment in this study. Crayfish were immunized by intramuscular injection of recombinant protein V28 (VP26 or VP24) at a dose of 2μg/g. The survival rate of crayfish immunized by VP28 showed a higher value than by VP26 or VP24 after WSSV challenge. Compared with the WSSV-positive control group, the VP28-immunized group could inhibit the replication of WSSV in crayfish, increasing the survival rate of crayfish to 66.67% after WSSV infection. The results of gene expression showed that VP28 treatment could enhance the expression of immune genes, mainly JAK and STAT genes. VP28 treatment also enhanced total hemocyte counts and enzyme activities including PO, SOD, and CAT in crayfish. VP28 treatment reduced the apoptosis of hemocytes in crayfish, as well as after WSSV infection. In conclusion, VP28 treatment can enhance the innate immunity of crayfish and has a significant effect on resistance to WSSV, and can be used as a preventive tool.

Duox mediated ROS production inhibited WSSV replication in Eriocheir sinensis under short-term nitrite stress

Nitrite stress and white spot syndrome virus (WSSV) infection are major problems threatening the sustainable and healthy development of Eriocheir sinensis. Some studies have found that nitrite stress can lead to the production of reactive oxygen species (ROS), whereas synthetic ROS plays a vital role in the signaling pathway. However, whether nitrite stress influences the infection of crabs by WSSV remains unclear. NADPH oxidases, including NOX1-5 and Duox1-2, are important for ROS production. In the present study, a novel Duox gene (designated as EsDuox) was identified from E. sinensis. The studies found that nitrite stress could increase the expression of EsDuox during WSSV infection and decrease the transcription of the WSSV envelope protein VP28. Moreover, nitrite stress could increase the production of ROS, and the synthesis of ROS relied on EsDuox. These results indicated a potential “nitrite stress–Duox activation–ROS production” pathway that plays a negative role in WSSV infection in E. sinensis. Further studies found that nitrite stress and EsDuox could promote the expression of EsDorsal transcriptional factor and antimicrobial peptides (AMPs) during WSSV infection. Moreover, the synthesis of AMPs was positively regulated by EsDorsal in the process of WSSV infection under nitrite stress. Furthermore, EsDorsal played an inhibitory role in the replication of WSSV under nitrite stress. Our study reveals a new pathway for “nitrite stress–Duox activation–ROS production–Dorsal activation–AMP synthesis” that is involved in the defense against WSSV infection in E. sinensis during short-term nitrite stress.

PcTrim prevents early infection with white spot syndrome virus by inhibiting AP1-induced endocytosis

Viruses have evolved various strategies to achieve early infection by initiating transcription of their own early genes via host transcription factors, such as NF-κb, STAT, and AP1. How the host copes with this immune escape has been a topic of interest. Tripartite motif (TRIM) family proteins with RING-type domains have E3 ubiquitin ligase activity and are known as host restriction factors. Trim has been reported to be associated with phagocytosis and is also believed to be involved in the activation of autophagy. Preventing the virus from entering the host cell may be the most economical way for the host to resist virus infection. The role of TRIM in the early stage of virus infection in host cells remains to be further interpreted. In the current study, a crayfish TRIM with a RING-type domain, designated as PcTrim, was significantly upregulated under white spot syndrome virus (WSSV) infection in the red swamp crayfish (Procambarus clarkii). Recombinant PcTrim significantly inhibited WSSV replication in crayfish. RNAi targeting PcTrim or blocking PcTrim with an antibody promoted WSSV replication in crayfish. Pulldown and co-IP assays showed that PcTrim can interact with the virus protein VP26. PcTrim restricts the expression level of dynamin, which is involved in the regulation of phagocytosis, by inhibiting AP1 entry into the nucleus. AP1-RNAi effectively reduced the expression levels of dynamin and inhibited host cell endocytosis of WSSV in vivo. Our study demonstrated that PcTrim might reduce early WSSV infection by binding to VP26 and then inhibiting AP1 activation, resulting in reduced endocytosis of WSSV in crayfish hemocytes.BjibKXcktbHxsdfgurhU3PVideo

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.