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. Video Abstract.

Invasion and Propagation of White Spot Syndrome Virus: Hijacking of the Cytoskeleton, Intracellular Transport Machinery, and Nuclear Import Transporters.

The pathogenesis of white spot syndrome virus (WSSV) is largely unclear. In this study, we found that actin nucleation and clathrin-mediated endocytosis were recruited for internalization of WSSV into crayfish hematopoietic tissue (Hpt) cells. This internalization was followed by intracellular transport of the invading virions via endocytic vesicles and endosomes. After envelope fusion within endosomes, the penetrated nucleocapsids were transported along microtubules toward the periphery of the nuclear pores. Furthermore, the nuclear transporter CqImportin α1/ß1, via binding of ARM repeat domain within CqImportin α1 to the nuclear localization sequences (NLSs) of viral cargoes and binding of CqImportin ß1 to the nucleoporins CqNup35/62 with the action of CqRan for docking to nuclear pores, was hijacked for both targeting of the incoming nucleocapsids toward the nuclear pores and import of the expressed viral structural proteins containing NLS into the cell nucleus. Intriguingly, dysfunction of CqImportin α1/ß1 resulted in significant accumulation of incoming nucleocapsids on the periphery of the Hpt cell nucleus, leading to substantially decreased introduction of the viral genome into the nucleus and remarkably reduced nuclear import of expressed viral structural proteins with NLS; both of these effects were accompanied by significantly inhibited viral propagation. Accordingly, the survival rate of crayfish post-WSSV challenge was significantly increased after dysfunction of CqImportin α1/ß1, also showing significantly reduced viral propagation, and was induced either by gene silencing or by pharmacological blockade via dietary administration of ivermectin per os. Collectively, our findings improve our understanding of WSSV pathogenesis and support future antiviral designing against WSSV. IMPORTANCE As one of the largest animal DNA viruses, white spot syndrome virus (WSSV) has been causing severe economical loss in aquaculture due to the limited knowledge on WSSV pathogenesis for an antiviral strategy. We demonstrate that the actin cytoskeleton, endocytic vesicles, endosomes, and microtubules are hijacked for WSSV invasion; importantly, the nuclear transporter CqImportin α1/ß1 together with CqRan were recruited, via binding of CqImportin ß1 to the nucleoporins CqNup35/62, for both the nuclear pore targeting of the incoming nucleocapsids and the nuclear import of expressed viral structural proteins containing the nuclear localization sequences (NLSs). This is the first report that NLSs from both viral structure proteins and host factor are elaborately recruited together to facilitate WSSV infection. Our findings provide a novel explanation for WSSV pathogenesis involving systemic hijacking of host factors, which can be used for antiviral targeting against WSSV disease, such as the blockade of CqImportin α1/ß1 with ivermectin.

Epigallocatechin-3-gallate inhibits replication of white spot syndrome virus in the freshwater crayfish Procambarus clarkii.

The freshwater crayfish Procambarus clarkii is native to North America and Mexico, and it was introduced to China in 1929. The production and consumption of P. clarkii in China are the highest worldwide, reaching 208.96 million tons in 2020. The white spot syndrome virus (WSSV) is a major pathogen that affects shrimp, crayfish, crabs and lobsters, and it has caused widespread loss to the P. clarkii industry. Epigallocatechin-3-gallate (EGCG), a small-molecule compound, has a multitude of biological functions and the ability to bind to the 37 kDa/67 kDa laminin receptor (LamR). EGCG has potential antiviral effects against WSSV. In this study, we evaluated the potential anti-WSSV applications of EGCG in P. clarkii. We demonstrated that various concentrations (10 µg/g·bw, 20 µg/g·bw and 40 µg/g·bw) of EGCG can suppress WSSV infection in P. clarkii. Histopathological examination revealed no characteristic pathological changes due to EGCG administration in P. clarkii tissues. Furthermore, pharmacokinetics studies of EGCG in P. clarkii revealed its rapid absorption (Tmax  = 2 h), and the peak concentrations of EGCG were 73.78 µg/g in the liver and 24.87 µg/g in the muscle. Our results indicate the high potential applications of EGCG against WSSV in P. clarkii.

Virome Analysis of Signal Crayfish (Pacifastacus leniusculus) along Its Invasion Range Reveals Diverse and Divergent RNA Viruses.

Crayfish are a keystone species of freshwater ecosystems and a successful invasive species. However, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of the invasive signal crayfish (Pacifastacus leniusculus) and to elucidate the potential differences in viral composition and abundance along its invasion range in the Korana River, Croatia. By the high-throughput sequencing of ribosomal RNA, depleted total RNA isolated from the crayfish hepatopancreas, and subsequent sequence data analysis, we identified novel and divergent RNA viruses, including signal crayfish-associated reo-like, hepe-like, toti-like, and picorna-like viruses, phylogenetically related to viruses previously associated with crustacean hosts. The patterns of reads abundance and calculated nucleotide diversities of the detected viral sequences varied along the invasion range. This could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density, the non-random dispersal of host individuals from the core to the invasion fronts, and the transfer of viruses from the native co-occurring and phylogenetically related crayfish species. The study reveals a high, previously undiscovered diversity of divergent RNA viruses associated with signal crayfish, and sets foundations for understanding the potential risk of virus transmissions as a result of this invader's dispersal.

Effects of dietary quercetin on the innate immune response and resistance to white spot syndrome virus in Procambarusclarkii.

In recent years, the use of natural products with immune-stimulating and antimicrobial properties has attracted increasing attention in aquaculture researches. In our study, the effect of diet supplemented with quercetin, a flavonoid commonly found in some types of plants substance on the innate immune response and disease resistance in crayfish (Procambarus clarkii) against white spot syndrome virus (WSSV) is reported. It was found that dietary 40 mg/kg quercetin significantly reduced the mortality of crayfish and WSSV copy number after WSSV challenge. Dietary quercetin increased catalase (CAT), and lysozyme (LZM) activity in crayfish. Dietary quercetin increased the expression of NF-κB, anti-lipopolysaccharide factor (ALF) and toll-like receptor (TLR) genes in crayfish. The apoptosis rate of hemocyte was increased by quercetin supplement in crayfish. Our results suggest that dietary quercetin may affect the innate immunity of crayfish and protect crayfish from WSSV infection.

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%, 100 mg/kg). Further, NAR, the main active compound of T. angustifolia, showed a much higher inhibition rate (92.85%, 50 mg/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.

The identification of a nuclear factor Akirin with regulating the expression of antimicrobial peptides in red swamp crayfish (Procambarus clarkii).

Akirin is a highly conserved nuclear factor among different species. It is closely related to skeletal muscle development, innate immune response, and tumorigenesis in a variety of animals. In invertebrates, Akirin is mainly involved in gene transcription and NF-κB dependent natural immune response. In the present study, a nuclear factor Akirin was identified from Procambarus clarkii. The Akirin protein of crayfish consists of 204 amino acids and is conserved among its family members, especially the nuclear localization signal peptide motif (KRRR). PcAkirin was highly expressed in stomach, intestines, and hepatopancreas. After A. hydrophila challenge, the transcription level of Akirin significantly increased in hemocyte and hepatopancreas. In addition, the recombinant Akirin protein was produced successfully and helpful to resist WSSV infection by increasing the expression level of some immune related genes. On the contrary, after interfering with Akirin gene by dsRNA, the crayfish increased the sensitivity to A. hydrophila and WSSV infections. The results are more obvious in the accumulated mortality of P. clarkii infected with A. hydrophila and WSSV. All these results suggested that Akirin played a significant role in innate immune responses and protected it from WSSV and bacterial infection in crayfish.

Patterns of infection in a native and an invasive crayfish across the UK.

Invasive crayfish and the introduction of non-native diseases pose a significant risk for the conservation of endangered, white-clawed crayfish (Austropotamobius pallipes). Continued pollution of waterways is also of concern for native species and may be linked with crayfish disease dynamics. We explore whether crayfish species or environmental quality are predictors of infection presence and prevalence in native A. pallipes and invasive signal crayfish (Pacifastacus leniusculus). We use a seven-year dataset of histology records, and a field survey comparing the presence and prevalence of infectious agents in three isolated A. pallipes populations; three isolated P. leniusculus populations, and three populations where the two species had overlapped in the past. We note a lower diversity of parasites (Simpson's Index) in P. leniusculus ('Pacifastacus leniusculus Bacilliform Virus' - PlBV) (n = 1 parasite) relative to native A. pallipes (n = 4 parasites), which host Thelohania contejeani, 'Austropotamobius pallipes bacilliform virus' (ApBV), Psorospermium haeckeli and Branchiobdella astaci, at the sites studied. The infectious group present in both species was an intranuclear bacilliform virus of the hepatopancreas. The prevalence of A. astaci in A. pallipes populations was higher in more polluted water bodies, which may reflect an effect of water quality, or may be due to increased chance of transmission from nearby P. leniusculus, a species commonly found in poor quality habitats.

Microbial pathogens of freshwater crayfish: A critical review and systematization of the existing data with directions for future research.

Despite important ecological role and growing commercial value of freshwater crayfish, their diseases are underresearched and many studies examining potential crayfish pathogens do not thoroughly address their epizootiology, pathology or biology. This study reviews over 100 publications on potentially pathogenic viruses, bacteria, fungi and fungal-like microorganisms reported in crayfish and systematizes them based on whether pathogenicity has been observed in an analysed species. Conclusions on pathogenicity were based on successful execution of infectivity trials. For 40.6% of examined studies, microbes were successfully systematized, while for more than a half (59.4%) no conclusion on pathogenicity could be made. Fungi and fungal-like microorganisms were the most studied group of microbes with the highest number of analysed hosts, followed by bacteria and viruses. Our analysis demonstrated the need for: (a) inclusion of higher number of potential host species in the case of viruses, (b) research of bacterial effects in tissues other than haemolymph, and (c) more research into potential fungal and fungal-like pathogens other than Aphanomyces astaci. We highlight the encountered methodological challenges and biases and call for a broad but standardized framework for execution of infectivity trials that would enable systematic data acquisition on interactions between microbes and the host.

Recent insights into anti-WSSV immunity in crayfish.

White spot syndrome virus (WSSV) is currently the most severely viral pathogen for farmed crustaceans such as shrimp and crayfish, which has been causing huge economic losses for crustaceans farming worldwide every year. Unfortunately, study on the molecular mechanisms of WSSV has been restricted by the lack of crustacean cell lines for WSSV propagation as well as the incompletely annotated genomes for host species, resulting in limited elucidation for WSSV pathogenesis at present. In addition to the findings of anti-WSSV response in shrimp, some of novel cellular events involved in WSSV infection have been recently revealed in crayfish, including endocytosis and intracellular transport of WSSV, innate immune pathways in response to WSSV infection, and regulation of viral gene expression by host genes. Despite these advances, many fundamental gaps in WSSV pathogenesis are still remaining, for example, how WSSV genome enters into nucleus and how the progeny virions are fully assembled in the host cell nucleus. In this review, recent findings in WSSV infection mechanism and the antiviral immunity against WSSV in crayfish are summarized and discussed, which may provide us a better understanding of the WSSV pathogenesis as well as new ideas for the target design of antiviral drugs against WSSV in crustaceans farming.

A novel Dicistro-like virus discovered in Procambarus clarkii with "Black May" disease.

The peak period of morbidity and death in cultured Procambarus clarkii is around May each year and is called the "Black May" disease. The pathogen causing "Black May" disease is believed to be a white spot syndrome virus (WSSV). In 2018, a significant number of P. clarkii died in the pond culture of Xinglong Township, Xuyi County. Two sampling tests on the affected pond showed that, in addition to WSSV, a novel Dicistro-like virus (PcDV) was present. Genomic sequence analysis indicated that this new virus belongs to the Dicistroviridae family, Picornaviridaes order. A high number of spherical particles were detected in gill tissues of P. clarkii with "Black May" disease by electron microscopy, a finding consistent with viruses from the Picornaviridaes order. From October 2018 to September 2019, we took monthly samples from Hubei, Jiangsu and Anhui provinces, and tested for the presence of PcDV and WSSV in P. clarkii. The detection rates of PcDV in P. clarkii peaked from April to June, consistent with the onset of the "Black May" disease. In conclusion, we believe that the discovery of PcDV will provide new research directions for investigating the pathogens causing "Black May" disease in P. clarkii.

CqPP2A inhibits white spot syndrome virus infection by up-regulating antimicrobial substances expression in red claw crayfish Cherax quadricarinatus.

Protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase, a highly conserved enzyme widely expressed in eukaryotic cells, which accounts for a majority of the serine/threonine phosphatase activity in cells implicated in regulation of immune signaling pathways and antiviral response. However, most of studies about PP2A have been conducted in mammals but few in crustaceans. In this study, two subunits of PP2A (named as CqPP2Ab and CqPP2Ac) were characterized to be involved in white spot syndrome virus (WSSV) infection in the haematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus. The open reading frame (ORF) of CqPP2Ab was 1341 bp encoding 446 amino acids with seven WD40 domains, and the ORF of CqPP2Ac was 930 bp encoding 309 amino acids with a PP2Ac domain. Tissue distribution analysis showed that the mRNA transcript of CqPP2Ab and CqPP2Ac were both widely expressed in all the tested tissues with the highest expression in hemocyte, followed by high expression in Hpt. The gene expressions of CqPP2Ab and CqPP2Ac were both significantly down-regulated at 6 h post WSSV infection (6 hpi) in Hpt cells. Importantly, the expression of viral immediate early gene IE1 and late viral gene envelope protein VP28 were both significantly increased post WSSV infection after gene silencing of CqPP2Ab or CqPP2Ac in Hpt cells, suggesting that CqPP2Ab and CqPP2Ac could inhibit WSSV infection in Hpt cells, probably by increasing the antimicrobial substances expression in consideration to the significantly reduced expression of anti-lipopolysaccharide factor, crustin, and lysozyme after gene silencing of CqPP2Ab or CqPP2Ac, respectively. These findings provide a new light on the mechanism of WSSV infection and the antiviral response in crustaceans.

White Spot Syndrome Virus Benefits from Endosomal Trafficking, Substantially Facilitated by a Valosin-Containing Protein, To Escape Autophagic Elimination and Propagate in the Crustacean Cherax quadricarinatus.

As the most severely lethal viral pathogen for crustaceans in both brackish water and freshwater, white spot syndrome virus (WSSV) has a mechanism of infection that remains largely unknown, which profoundly limits the control of WSSV disease. By using a hematopoietic tissue (Hpt) stem cell culture from the red claw crayfish Cherax quadricarinatus suitable for WSSV propagation in vitro, the intracellular trafficking of live WSSV, in which the acidic-pH-dependent endosomal environment was a prerequisite for WSSV fusion, was determined for the first time via live-cell imaging. When the acidic pH within the endosome was alkalized by chemicals, the intracellular WSSV virions were detained in dysfunctional endosomes, resulting in appreciable blocking of the viral infection. Furthermore, disrupted valosin-containing protein (C. quadricarinatus VCP [CqVCP]) activity resulted in considerable aggregation of endocytic WSSV virions in the disordered endosomes, which subsequently recruited autophagosomes, likely by binding to CqGABARAP via CqVCP, to eliminate the aggregated virions within the dysfunctional endosomes. Importantly, both autophagic sorting and the degradation of intracellular WSSV virions were clearly enhanced in Hpt cells with increased autophagic activity, demonstrating that autophagy played a defensive role against WSSV infection. Intriguingly, most of the endocytic WSSV virions were directed to the endosomal delivery system facilitated by CqVCP activity so that they avoided autophagy degradation and successfully delivered the viral genome into Hpt cell nuclei, which was followed by the propagation of progeny virions. These findings will benefit anti-WSSV target design against the most severe viral disease currently affecting farmed crustaceans.IMPORTANCE White spot disease is currently the most devastating viral disease in farmed crustaceans, such as shrimp and crayfish, and has resulted in a severe ecological problem for both brackish water and freshwater aquaculture areas worldwide. Efficient antiviral control of WSSV disease is still lacking due to our limited knowledge of its pathogenesis. Importantly, research on the WSSV infection mechanism is also quite meaningful for the elucidation of viral pathogenesis and virus-host coevolution, as WSSV is one of the largest animal viruses, in terms of genome size, that infects only crustaceans. Here, we found that most of the endocytic WSSV virions were directed to the endosomal delivery system, strongly facilitated by CqVCP, so that they avoided autophagic degradation and successfully delivered the viral genome into the Hpt cell nucleus for propagation. Our data point to a virus-sorting model that might also explain the escape of other enveloped DNA viruses.

WSSV proteins and DNA genome released by ultrasonic rupture can infect crayfish as effectively as intact virions.

Proteins and nucleic acids from ultrasonically ruptured white spot syndrome virus (WSSV) can infect crayfish and cause death as effectively as intact WSSV virions. In this study, ultrasound was used to rupture the virus and the resulting suspension was filtered through a 50 nm membrane. Analysis by PCR and SDS-PAGE showed that both viral genes (VP19, VP26, VP28 and DNA polymerase) and proteins (VP15, VP19, VP26 and VP28) were present in the filtered solution. Electron microscopy showed that there were no intact virions in the filtered solution. When crayfish were injected with the filtered solution or with intact WSSV, the mortality in each group was 100 %. The same result was seen when crayfish were challenged orally with the filtered solution and intact WSSV. The filtered solution of ultrasonically ruptured virus, which contains viral proteins and residual DNA genome, can thus infect the host as effectively as intact virions. When the solution of viral proteins and residual DNA genome was digested with DNase I and then injected into crayfish, the survival rate was 100 %. We also found that, although viral proteins (except VP15) in the solution of ruptured virus were destroyed by treatment with DNase I, DNase I did not destroy the structural proteins of intact virions. A remaining viral protein in the DNase I-treated solution protects the DNA genome from degradation and we concluded that this protein is VP15, which is a DNA-binding protein. Our study highlights the extreme danger in producing vaccines from proteins obtained by ultrasonic rupture of viruses sincethe viral DNA genome is difficult to degrade and, if present, will lead to viral infection.

Environmental concentrations of sulfamethoxazole increase crayfish Pacifastacus leniusculus susceptibility to White Spot Syndrome Virus.

Antibiotics used for humans and livestock are emerging as pollutants in aquatic environments. However, little is known about their effect on aquatic organisms, especially in crustaceans. In the present study, the freshwater crayfish Pacifastacus leniusculus was exposed during 21 days to environmental concentrations of sulfamethoxazole (SMX) (100 ng/L and 1 µg/L). Subsequently, the crayfish susceptibility to infection was evaluated by using White Spot Syndrome Virus (WSSV) challenge, a well-known crustacean pathogen. The median survival time of the infected crayfish exposed to 100 ng/L SMX was one day, whereas the control and the group exposed to 1 µg/L SMX survived for two and three days, respectively. In order to elucidate the effect of SMX upon the crayfish immune response, new sets of crayfish were exposed to the same SMX treatments to evaluate mRNA levels of immune-related genes which are expressed and present in hemocytes and intestine, and to perform total and differential hemocyte counts. These results show a significant down-regulation of the antimicrobial peptide (AMP) Crustin 3 in hemocytes from the 100 ng/L SMX group, as well as a significant up-regulation of the AMP Crustin 1 in intestines from the 1 µg/L SMX group. Semigranular and total hemocyte cell number were observed to be significantly lower after exposure to 100 ng/L SMX in comparison with the control group. The present study demonstrates that environmentally relevant SMX concentrations in the water at 100 ng/L led to an increased WSSV susceptibility, that may have been caused by a reduction of circulating hemocytes. Nevertheless, SMX concentrations of 1 µg/L could marginally and for a few days have an immunostimulatory effect.

Dietary Hizikia fusiforme enhance survival of white spot syndrome virus infected crayfish Procambarus clarkii.

The sea vegetable Hizikia fusiforme is not only a good source of dietary fiber but also enhances immunity. In this study, we investigated the effects of H. fusiforme on innate immunity in invertebrates, using white spot syndrome virus (WSSV) challenge in the crayfish, Procambarus clarkii. Supplementation with H. fusiforme significantly reduced mortality caused by WSSV infection and also reduced copy numbers of the WSSV protein VP28. Quantitative reverse transcription-polymerase chain reaction showed that supplementation of feed with H. fusiforme increased the expression of immune-related genes, including NF-κB and crustin 1. Further analysis showed that supplementation with H. fusiforme also affected three immune parameters, total hemocyte count, and phenoloxidase and superoxide dismutase activity. H. fusiforme treatment significantly increased hemocyte apoptosis rates in both WSSV-infected and uninfected crayfish. H. fusiforme thus regulates the innate immunity of crayfish, and both delays and reduces mortality after WSSV challenge. Our study demonstrates the potential for the commercial use of H. fusiforme, either therapeutically or prophylactically, to regulate the innate immunity and protect crayfish against WSSV infection.

The red swamp crayfish, Procambarus clarkii cathepsin C, participates in the innate immune response to the viral and bacterial pathogens.

The cathepsin C, a lysosomal cysteine protease, involves the modulation of immune and inflammatory responses in living organisms. However, the knowledge on cathepsin C in red swamp crayfish (Procambarus clarkii), a freshwater crustacean with economic values, remained unclear. In the present study, we provide identification and molecular characterization of cathepsin C from P. clarkii. (Hereafter Pc-cathepsin C). The Pc-cathepsin C cDNA contained a 1356 bp open reading frame that encoded a protein of 451 amino acid residues. The deduced amino acid sequence comprised of cathepsin C exclusion domain and pept_C1 domain, and also catalytic residues (Cys248, His395 and Asn417). Analysis of the transcriptional patterns of the Pc-cathepsin C gene revealed that it was broadly distributed in various tissues of P. clarkii, and it was more abundant in the hepatopancreas and gut. Following a challenge with viral and bacterial pathogen-associated molecular patterns, the expression of Pc-cathepsin C was strongly enhanced at different time points. The knockdown of Pc-cathepsin C, altered the expression of immune-responsive genes, suggesting its immunoregulatory role in P. clarkii. This study has identified and provided the immunoregulatory function of Pc-cathepsin C, which will contribute to further investigation of the molecular mechanism of cathepsin C in crustaceans.

A negative elongation factor E inhibits white spot syndrome virus replication by suppressing promoter activity of the viral immediate early genes in red claw crayfish Cherax quadricarinatus.

Invertebrates rely solely on the innate immune system to protect against virus infection, while the viral infection must rely on the transcriptional system of the host cell to achieve the expression of viral genes, which is naturally regulated by the host's transcriptional system. However, the mechanism of the host against viral transcription in host cells is still poorly understood in crustaceans. Previously, we found that the partial transcript sequence of a negative elongation factor E (named as CqNELF-E) was up-regulated in a differentially expressed transcriptome library of the haematopietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus upon white spot syndrome virus (WSSV) infection, suggesting a possible role of CqNELF-E in WSSV-host interaction. In the present study, we revealed the function of CqNELF-E. The full-length cDNA sequence of CqNELF-E was identified with 1726 bp from red claw crayfish, which contained an open reading frame of 816 bp, encoding 271 amino acids. Amino acid sequencing analysis revealed that the CqNELF-E had a conserved RNA recognition motif (RRM) and a leucine zipper motif (LZM). Tissue distribution analysis showed that CqNELF-E was widely expressed in various tissues with the highest expression in muscle, relatively abundant in Hpt and the lowest presence in heart. Interestingly, the gene expression of CqNELF-E was significantly up-regulated at both 6 and 12 hpi after WSSV infection in Hpt cell cultures in red claw crayfish. In addition, the expression of both the viral immediately early gene (IE) 1 (IE1) and a late gene envelope protein VP28 were significantly increased after gene silencing of CqNELF-E in Hpt cells, indicating the potential suppression role of CqNELF-E against the viral infection. Further study revealed that the CqNELF-E had an inhibitory effect on the promoter activity of WSSV IE genes WSV051, WSV069 (IE1) and WSV083 by a dual luciferase reporter gene assay. Taken together, these results suggest that CqNELF-E plays an antiviral role, probably via inhibition on the viral transcription activity in WSSV infection in a crustacean.

Molecular cloning and functional analysis of three STAT isoforms in red swamp crayfish Procambarus clarkii.

The Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway is associated with the innate immune system and plays crucial roles in the mediation of immune response to viral infections. In this study, three STAT isoform cDNAs were cloned from the red swamp crayfish Procambarus clarkii, and they were designated as PcSTATa, PcSTATb, and PcSTATc. PcSTATa and PcSTATb were generated through the alternative splicing of the last exon, and PcSTATc was produced by intron retention. PcSTATa, PcSTATb, and PcSTATc contained 2382, 2337, and 2274 bp open reading frames encoding proteins with 793, 778, and 757 amino acid residues, respectively. Domain prediction analysis revealed that three isoforms of PcSTATs contain a STAT interaction domain, a STAT all-alpha domain, a STAT DNA binding domain, and a Src-homology 2 domain. The mRNA transcripts of three PcSTAT isoforms were detected in all examined tissues of male and female crayfish. The expression levels of the three PcSTAT isoforms in the hemocytes, gills, and intestines significantly changed after the white spot syndrome virus (WSSV) challenge. PcSTAT silencing by dsRNA interference could positively regulate the expression levels of three anti-lipopolysaccharide factors (PcALF1, PcALF2, and PcALF6) and two crustins (PcCrus1 and PcCrus2) and negatively regulate the expression levels of three ALFs (PcALF3, PcALF4, and PcALF5) and two crustins (PcCrus3 and PcCrus4). These results suggest that all three PcSTAT isoforms are involved in the host defense against WSSV infection.