Insect cells of Spodoptera frugiperda support WSSV gene replication but not progeny virion assembly.

The lacking of stable and susceptible cell lines has hampered research on pathogenic mechanism of crustacean white spot syndrome virus (WSSV). To look for the suitable cell line which can sustain WSSV infection, we performed the studies on WSSV infection in the Spodoptera frugiperda (Sf9) insect cells. In consistent with our previous study in vitro in crayfish hematopoietic tissue cells, the WSSV envelope was detached from nucleocapsid around 2 hpi in Sf9 cells, which was accompanied with the cytoplasmic transport of nucleocapsid toward the cell nucleus within 3 hpi. Furthermore, the expression profile of both gene and protein of WSSV was determined in Sf9 cells after viral infection, in which a viral immediate early gene IE1 and an envelope protein VP28 exhibited gradually increased presence from 3 to 24 hpi. Similarly, the significant increase of WSSV genome replication was found at 3-48 hpi in Sf9 cells after infection with WSSV, indicating that Sf9 cells supported WSSV genome replication. Unfortunately, no assembled progeny virion was observed at 24 and 48 hpi in Sf9 cell nuclei as determined by transmission electron microscope, suggesting that WSSV progeny could not be assembled in Sf9 cell line as the viral structural proteins could not be transported into cell nuclei. Collectively, these findings provide a cell model for comparative analysis of WSSV infection mechanism with crustacean cells.

The crustacean DNA virus tegument protein VP26 binds to SNAP29 to inhibit SNARE complex assembly and autophagic degradation.

Autophagy generally functions as a cellular surveillance mechanism to combat invading viruses, but viruses have evolved various strategies to block autophagic degradation and even subvert it to promote viral propagation. White spot syndrome virus (WSSV) is the most highly pathogenic crustacean virus, but little is currently known about whether crustacean viruses such as WSSV can subvert autophagic degradation for escape. Here, we show that even though WSSV proliferation triggers the accumulation of autophagosomes, autophagic degradation is blocked in the crustacean species red claw crayfish. Interestingly, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex including CqSNAP29, CqVAMP7, and the novel autophagosome SNARE protein CqSyx12 is required for autophagic flux to restrict WSSV replication, as revealed by gene silencing experiments. Simultaneously, the expressed WSSV tegument protein VP26, which likely localizes on autophagic membrane mediated by its transmembrane region, binds the Qb-SNARE domain of CqSNAP29 to competitively inhibit the binding of CqSyx12-Qa-SNARE with CqSNAP29-Qb-SNARE; this in turn disrupts the assembly of the CqSyx12-SNAP29-VAMP7 SNARE complex, which is indispensable for the proposed fusion of autophagosomes and lysosomes. Consequently, the autophagic degradation of WSSV is likely suppressed by the expressed VP26 protein in vivo in crayfish, thus probably protecting WSSV components from degradation via the autophagosome-lysosome pathway, resulting in evasion by WSSV. Collectively, these findings highlight how a DNA virus can subvert autophagic degradation by impairing the assembly of the SNARE complex to achieve evasion, paving the way for understanding host-DNA virus interactions from an evolutionary point of view, from crustaceans to mammals.IMPORTANCEWhite spot syndrome virus (WSSV) is one of the largest animal DNA viruses in terms of its genome size and has caused huge economic losses in the farming of crustaceans such as shrimp and crayfish. Detailed knowledge of WSSV-host interactions is still lacking, particularly regarding viral escape from host immune clearance. Intriguingly, we found that the presence of WSSV-VP26 might inhibit the autophagic degradation of WSSV in vivo in the crustacean species red claw crayfish. Importantly, this study is the first to show that viral protein VP26 functions as a core factor to benefit WSSV escape by disrupting the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which is necessary for the proposed fusion of autophagosomes with lysosomes for subsequent degradation. These findings highlight a novel mechanism of DNA virus evasion by blocking SNARE complex assembly and identify viral VP26 as a key candidate for anti-WSSV targeting.

Kaposiform hemangioendothelioma presented with raynaud phenomenon: a case report.

BACKGROUND: Kaposiform hemangioendothelioma (KHE) is a rare vascular neoplasm affecting infants or young children. KHE includes a spectrum of lesions, ranging from small and superficial tumors to large and invasive lesions with Kasabach-Merritt phenomenon (KMP). Currently, no published studies have reported a KHE presenting as thrombocytopenia and Raynaud phenomenon. CASE PRESENTATION: A 2-year-old boy with right hand swelling and thrombocytopenia was admitted to our hospital. His right hand turned swelling and red, even occasionally cyanotic. This condition became worse in response to cool environments and improved with warming, and platelet counts were between 50 ~ 80 × 10^9/L. Physical examination on admission revealed the swelling and frostbite-like rash of the right-hand fingers, and the skin temperature of the right hand was lower than the left. On day 3 of admission, chest CT results showed an irregular mass on the right side of the spine. The puncture biopsy demonstrated positive CD31, D2-40, and FLI1 immunohistochemical staining, but negative GLUT1 staining, confirming the diagnosis of KHE. Furthermore, endothelin-1 (ET1) expression levels significantly increased, and eNOS and A20 expression levels significantly decreased comparing with control patients. The patient received methylprednisolone and sirolimus treatments, and his condition gradually improved during the follow-up. CONCLUSIONS: We reported the first case of KHE presenting with thrombocytopenia and Raynaud phenomenon. The development of Raynaud phenomenon could be associated with increased ET-1 and reduced eNOS and A20 expressions. Careful differential diagnosis of hidden KHE should be considered in children with thrombocytopenia and Raynaud phenomenon.

Decreased ubiquitin modifying enzyme A20 associated with hyper-responsiveness to ovalbumin challenge following intrauterine growth restriction.

BACKGROUND: Intrauterine growth restriction (IUGR) is strongly correlated with an increased risk of asthma later in life. Farm dust protects mice from developing house dust mite-induced asthma, and loss of ubiquitin modifying enzyme A20 in lung epithelium would abolish this protective effect. However, the mechanisms of A20 in the development of asthma following IUGR remains unknown. METHODS: An IUGR rat model induced by maternal nutrient restriction was used for investigating the role of A20 in the response characteristics of IUGR rats to ovalbumin (OVA) challenge. The ubiquitination of proteins and N6-methyladenosine (m6A) modifications were used to further assess the potential mechanism of A20. RESULTS: IUGR can reduce the expression of A20 protein in lung tissue of newborn rats and continue until 10 weeks after birth. OVA challenging can increase the expression of A20 protein in lung tissue of IUGR rats, but its level was still significantly lower than the control OVA group. The differentially ubiquitinated proteins in lung tissues were also observed in IUGR and normal newborn rats. Furthermore, this ubiquitination phenomenon continued from the newborn to adulthood. In the detected RNA methylations, m6A abundance of the motif GGACA was the highest. The higher abundances of m6A modification of A20 mRNA from IUGR were negatively correlated with the trend of A20 protein levels. CONCLUSION: These findings indicate A20 as a key regulator during the development of asthma following IUGR, providing further insight into the prevention of asthma induced by environmental factors.

Autophagy and white spot syndrome virus infection in crustaceans.

Autophagy is an evolutionarily conserved process of degradation in eukaryotes, which can form double-membrane vesicles for delivering the trapped cargo to lysosome for degradation, also facilitate host cells against the invasion of foreign pathogens. Recently, autophagy was reported to participate in viral infection in crustaceans. White spot syndrome virus (WSSV) is the most severely viral pathogen for farmed crustaceans, particularly in crayfish and shrimp. In this review, we summarized and discussed the current findings of autophagy involved in WSSV infection in crustaceans, particularly focusing on the identified autophagy-related molecules and their effects on viral infection. We hope this summary will provide us a better understanding of autophagy and its contribution to antiviral immunity in crustaceans.

A Deacetylase CqSIRT1 Promotes WSSV Infection by Binding to Viral Envelope Proteins in Cherax quadricarinatus.

Sirtuin 1 (SIRT1), a member of the class III lysine deacetylases, exhibits powerful functional diversity in physiological processes and disease occurrences. However, the potential molecular mechanism underlying the role of SIRT1 during viral infection in crustaceans is poorly understood. Herein, SIRT1 was functionally characterized from the red claw crayfish Cherax quadricarinatus, which possesses typically conserved deacetylase domains and strong evolutionary relationships across various species. Moreover, gene knockdown of CqSIRT1 in crayfish haematopoietic tissue (Hpt) cell culture inhibited white spot syndrome virus (WSSV) late envelope gene vp28 transcription. In contrast, enhancement of deacetylase activity using a pharmacological activator promoted the replication of WSSV. Mechanically, CqSIRT1 was co-localized with viral envelope protein VP28 in the nuclei of Hpt cells and directly bound to VP28 with protein pulldown and co-immunoprecipitation assays. Furthermore, CqSIRT1 also interacted with another two viral envelope proteins, VP24 and VP26. To the best of our knowledge, this is the first report that WSSV structural proteins are linked to lysine deacetylases, providing a better understanding of the role of CqSIRT1 during WSSV infection and novel insights into the basic mechanism underlying the function of lysine deacetylases in crustaceans.

Metabolomics of bronchoalveolar lavage in children with persistent wheezing.

BACKGROUND: Recent studies have demonstrated the important role of metabolomics in the pathogenesis of asthma. However, the role of lung metabolomics in childhood persistent wheezing (PW) or wheezing recurrence remains poorly understood. METHODS: In this prospective observational study, we performed a liquid chromatography/mass spectrometry-based metabolomic survey on bronchoalveolar lavage samples collected from 30 children with PW and 30 age-matched infants (control group). A 2-year follow-up study on these PW children was conducted. RESULTS: Children with PW showed a distinct characterization of respiratory metabolome compared with control group. Children with PW had higher abundances of choline, oleamide, nepetalactam, butyrylcarnitine, L-palmitoylcarnitine, palmitoylethanolamide, and various phosphatidylcholines. The glycerophospholipid metabolism pathway was the most relevant pathway involving in PW pathophysiologic process. Additionally, different gender, prematurity, and systemic corticoids use demonstrated a greater impact in airway metabolite compositions. Furthermore, for PW children with recurrence during the follow-up period, children who were born prematurely had an increased abundance of butyrylcarnitine relative to those who were carried to term. CONCLUSIONS: This study suggests that the alterations of lung metabolites could be associated with the development of wheezing, and this early alteration could also be correlated with wheezing recurrence later in life.

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.

Etiologic Profile of Older Children With Diffuse Radiological Changes in Eastern China.

Objective: To analyze the etiology of chest diffuse radiological changes (DRC) in children older than 2 years. Methods: A retrospective study was conducted on a primary cohort of children with DRC underwent high resolution computed tomography (HRCT). Results: DRC mainly included bronchial wall thickening, interlobular septal thickening, pleural thickening, ground glass opacity, mosaic perfusion, reticular & linear opacities, nodular opacity, and tree-in-bud. Of the identified 457 children with DRC, 83 of children older than 2 years with DRC were included in the present study. Ground glass opacity (53, 63.9%) and reticular & linear opacities (44, 53.0%) were frequently identified findings of HRCT, and no tree-in-bud pattern was observed. By contrast, among children with DRC by M. pneumoniae (n = 64), bronchial wall thickening (33, 51.6%), and mosaic perfusion (17, 26.6%) were common patterns of HRCT in addition to ground glass opacity (36, 56.3%). Most of etiologies were connective tissue disease (24, 28.9%), followed by diffuse alveolar hemorrhage syndrome (9, 10.8%), Langerhans cell histiocytosis (7, 8.4%), and recurrent aspiration (6, 7.2%). Conclusions: This study adds further insights into the role of HRCT in diagnosing childhood interstitial lung diseases, indirectly reflecting disease compositions.

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.

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.

A barrier-to-autointegration factor promotes white spot syndrome virus infection in a crustacean Cherax quadricarinatus.

Barrier-to-autointegration factor (BAF) is a highly conserved DNA binding protein that participates in a variety of biological processes such as transcription, epigenetic regulation and antiviral immunity in vertebrates. However, the function of BAF is poorly understood in crustaceans. In this study, we identified a barrier-to-autointegration factor (CqBAF) from red claw crayfish Cherax quadricarinatus, which was responsive to white spot syndrome virus (WSSV) infection. The full-length cDNA sequence of CqBAF was 544 bp, including an open reading frame of 273 bp encoding 90 amino acids, a 107 bp of 5'-Untranslated Regions (5'-UTR) and a 164 bp of 3'-UTR. Gene expression analysis showed that CqBAF was distributed in all tissues examined with the highest expression in the crayfish haematopietic tissue (Hpt), which protein expression was also significantly up-regulated by WSSV infection in Hpt cells. Furthermore, the transcripts of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV were clearly reduced in Hpt cells after gene silencing of CqBAF. Importantly, the promoter activity of two immediate early genes of WSSV, including WSV051 and IE1, was strongly enhanced by the increased phosphorylation of CqBAF, which also facilitated the accumulation of CqBAF protein in the cytoplasm of Sf9 cells. Taken together, these data suggest that CqBAF is likely to increase the replication of WSSV by promoting the transcription of viral immediate early genes, probably regulated by phosphorylation of CqBAF, which sheds new light on the molecular mechanism of WSSV infection.

A cytokine receptor domeless promotes white spot syndrome virus infection via JAK/STAT signaling pathway in red claw crayfish Cherax quadricarinatus.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is pivotal in immune responses for a variety of pathogens in both vertebrates and invertebrates. Domeless (Dome), as a unique cytokine receptor, involves in the upstream JAK/STAT pathway in invertebrates. In this study, the full-length cDNA sequence of a cytokine receptor Dome was identified from red claw crayfish Cherax quadricarinatus (named as CqDome), which contained an open reading frame of 4251 bp, encoding 1416 amino acids. The CqDome contained extracellular conservative domains of a signal peptide, two cytokine binding modules (CBM), three fibronectin-type-III-like (FN3) domains and a transmembrane region. Tissue distribution analysis showed that CqDome generally expressed in all the tissues selected with a high expression in hemocyte. The gene expression of both the viral immediately early gene (IE1) and a late gene envelope protein VP28 of white spot syndrome virus (WSSV) were significantly decreased after gene silencing of CqDome in crayfish haematopoietic tissue (Hpt) cells, indicating a key role of CqDome in promoting WSSV infection. Furthermore, the phosphorylation level of CqSTAT was significantly inhibited by gene silencing of CqDome in Hpt cells, indicating that CqDome participated in signal transduction of JAK/STAT pathway in red claw crayfish. These data together suggest that CqDome is likely to promote WSSV infection via JAK/STAT pathway, which sheds new light on further elucidation of the pathogenesis of WSSV.

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.

A histone K-lysine acetyltransferase CqKAT2A-like gene promotes white spot syndrome virus infection by enhancing histone H3 acetylation in red claw crayfish Cherax quadricarinatus.

In contrast to that hypoacetylation of histones is associated with condensed chromatin and gene silencing, the hyperacetylation of histones can promote an "open chromatin" conformation and transcriptional activation, which is recruited by some viruses to enhance the viral genome replication in host cells. However, the function of histone acetylation modification in the infection of white spot syndrome virus (WSSV), one of the most virulent pathogens for crustaceans like shrimp and crayfish at present, is still unknown. Previously, we found that the transcript of a histone K-Lysine acetyltransferase CqKAT2A-like gene was down-regulated in a differentially expressed transcriptome library of the haematopietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus upon WSSV infection at 12 hpi. To further reveal its possible role in anti-WSSV response, CqKAT2A-like gene was then identified with an open reading frame (ORF) of 2523 bp encoding 840 amino acids, which contained a conserved PCAF-N domain, acetyltransf1 domain and bromo domain. Gene expression analysis showed that CqKAT2A-like was distributed in all tissues examined with high presence in haemocyte and muscle, and the transcript was significantly down-regulated after WSSV infection in Hpt cells. Furthermore, the level of histone H3 acetylation (H3ac) was strongly reduced by gene silencing of CqKAT2A-like, which was accompanied with the significantly decreased gene expression of WSSV in Hpt cells, suggesting that CqKAT2A-like gene can promote the activity H3ac and the replication of WSSV. When the H3ac was induced by histone deacetyltransferase inhibitor TSA, the transcription of WSSV genes including both IE1 and VP28 genes was significantly increased, indicating that H3ac participated in WSSV infection in Hpt cells. Taken together, these data suggest that CqKAT2A-like gene might promote the replication of WSSV by regulating H3ac, which sheds new light on the pathogenesis of WSSV in crustaceans.

An Ns1abp-like gene promotes white spot syndrome virus infection by interacting with the viral envelope protein VP28 in red claw crayfish Cherax quadricarinatus.

Influenza A virus non-structural-1A binding protein (named as Ns1abp) was originally identified as a host protein from human that bound to the viral NS-1 protein. In our previous study, the expression of an Ns1abp-like gene (denoted as CqNs1abp-like gene) was found to be up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To elucidate the role of CqNs1abp-like gene involved in WSSV infection, we cloned the CqNs1abp-like gene in which the open reading frame was 2232 bp, encoding 743 amino acids with two typical domains of one BTB (Broad-Complex, Tramtrack and Bric a brac) domain at N-terminal and six Kelch domains at C-terminal. The gene expression profile showed that the mRNA transcript of CqNs1abp-like gene was widely expressed in all the tested tissues with highest expression in nerve, relatively high expression in Hpt and lowest expression in eyestalk. Importantly, both the WSSV entry and the viral replication were significantly reduced in Hpt cells after gene silencing of CqNs1abp-like gene. By using protein pull-down assay, we found that the recombinant BTB domain, six Kelch domains and CqNs1abp-like intact protein were all bound to the WSSV envelope protein VP28, respectively, in which the BTB domain showed slightly less binding affinity than that of the six Kelch domains or the recombinant intact protein. Besides, the WSSV entry into Hpt cells was clearly decreased when the virus was pre-incubated with the recombinant BTB domain, six Kelch domains, or the recombinant CqNs1abp-like intact protein, respectively, suggesting that the CqNs1abp-like gene was likely to function as a putative recognition molecular towards WSSV infection in a crustacean C. quadricarinatus. Taken together, these data shed new light on the mechanism of WSSV infection and a putatively novel target on anti-WSSV infection in crustacean farming.

A laminin-receptor-like protein regulates white spot syndrome virus infection by binding to the viral envelope protein VP28 in red claw crayfish Cherax quadricarinatus.

White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, which has been causing huge economic losses in global aquaculture. Laminin receptor (LR) is a cell surface receptor which participates in the interactions between cells as well as cells and extracellular matrix. Previously, we found that a CqLR-like gene was responsive to WSSV infection in the hematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus. To further reveal the role of CqLR-like gene involved in WSSV infection, the full-length cDNA of CqLR-like gene was cloned with 1000 bp, and the open reading frame encoded 308 amino acids with a conserved laminin-binding domain. Importantly, both the WSSV entry and viral replication were strongly reduced in Hpt cells after loss-of-function of CqLR-like gene by gene silencing. Protein interaction assay demonstrated that the recombinant CqLR-like protein could bind to WSSV virion in vitro by enzyme-linked immunosorbent assay and the binding affinity was in a dose-dependent manner. Furthermore, recombinant CqLR-like protein was found to bind to WSSV envelop protein VP28, but not other envelop proteins tested including VP19, VP24, and VP26, by pull down assay in HEK293T cells. In regarding to that LR is mainly localized on many types of cells' membrane, these data together suggested that CqLR-like protein was likely to function as a putative recognition molecule towards WSSV and act in the viral entry into a crustacean host cell, which may benefit the elucidation of the WSSV pathogenesis and further the pharmaceutical target for the possibly effective control of WSSV disease.