Abstract:
The white spot syndrome virus (WSSV) has been considered a serious threat to shrimp aquaculture. Besides, the activation of cell metabolism as an immune reaction to the virus is now recognized as a piece of the pivotal puzzle of the antiviral responses. Hence, this study explores the relationship between metabolic gene expression and antiviral responses in shrimp using transcriptome analysis. The RNA-seq libraries of Fenneropenaeus merguensis hemocytes after WSSV challenge at early (6 hpi) and late (24 hpi) stages of infection were analyzed to identify differentially expressed genes (DEGs) that the WSSV subverted the expression. One-hundred-thirty-three DEGs that were expressed in response to WSSV infection at both stages were identified. Based on the GO annotation, they were related to innate immunity and metabolic pathway. The expression correlation between “full term” (NGS) and qRT-PCR of 16 representative DEGs is shown. Noticeably, the expression profiles of all the selected metabolic genes involved in glucose metabolism, lipid metabolism, amino acid metabolism, and nucleotide metabolism showed a specific correlation between NGS and qRT-PCR upon WSSV infection. Of these, we further characterized the function related to the WSSV response of glutamine: fructose-6-phosphate aminotransferase (FmGFAT), the rate-limiting enzyme of the hexosamine biosynthesis pathway, which was found to be up-regulated at the late stage of WSSV infection. Suppression of FmGFAT by RNA interference resulted in postponing the death of WSSV-infected shrimp and reduction of viral copy number. These results suggested that the FmGFAT is linked between metabolic change and WSSV responses in shrimp, where the virus-induced metabolic rewiring hijack biological compounds and/or energy sources to benefit the viral replication process. © 2021 Elsevier Ltd

Keyword: Fenneropenaeus merguiensis; Glutamine: fructose-6-phosphate aminotransferase; Immunometabolism-related genes; Transcriptome profiling; White spot syndrome virus

Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85118848292&doi=10.1016%2fj.fsi.2021.11.006&partnerID=40&md5=a74c8de9ad99dd55424b71a9e4c2a9b0

DOI: 10.1016/j.fsi.2021.11.006

Abstract:
Host-specific legume-rhizobium symbiosis is strictly controlled by rhizobial type III effectors (T3Es) in some cases. Here, we demonstrated that the symbiosis of Vigna radiata (mung bean) with Bradyrhizobium diazoefficiens USDA110 is determined by NopE, and this symbiosis is highly dependent on host genotype. NopE specifically triggered incompatibility with V. radiata cv. KPS2, but it promoted nodulation in other varieties of V. radiata, including KPS1. Interestingly, NopE1 and its paralogue NopE2, which exhibits calcium-dependent autocleavage, yield similar results in modulating KPS1 nodulation. Furthermore, NopE is required for early infection and nodule organogenesis in compatible plants. Evolutionary analysis revealed that NopE is highly conserved among bradyrhizobia and plant-associated endophytic and pathogenic bacteria. Our findings suggest that V. radiata and B. diazoefficiens USDA110 may use NopE to optimize their symbiotic interactions by reducing phytohormone-mediated ETI-type (PmETI) responses via salicylic acid (SA) biosynthesis suppression. © 2021, The Author(s).

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Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85112689107&doi=10.1038%2fs41598-021-95925-4&partnerID=40&md5=7e78b3f6a0cdc42451048182307f913e

DOI: 10.1038/s41598-021-95925-4

Abstract:
MicroRNAs (miRNAs) suppress gene expression and regulate biological processes. Following small RNA sequencing, shrimp hemocytes miRNAs differentially expressed in response to acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus (VPAHPND) were discovered and some were confirmed by qRT-PCR. VPAHPND-responsive miRNAs were predicted to target several genes in various immune pathways. Among them, lva-miR-4850 is of interest because its predicted target mRNAs are two important genes of the proPO system; proPO2 (PO2) and proPO activating factor 2 (PPAF2). The expression of lva-miR-4850 was significantly decreased after VPAHPND infection, whereas those of the target mRNAs, PO2 and PPAF2, and PO activity were significantly upregulated. Introducing the lva-miR-4850 mimic into VPAHPND-infected shrimps caused a reduction in the PO2 and PPAF2 transcript levels and the PO activity, but significantly increased the number of bacteria in the VPAHPND targeted tissues. This result inferred that lva-miR-4850 plays a crucial role in regulating the proPO system via suppressing expression of PPAF2 and PO2. To fight against VPAHPND infection, shrimp downregulated lva-miR-4850 expression resulted in proPO activation. © 2021, The Author(s).

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Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85101466540&doi=10.1038%2fs41598-021-82881-2&partnerID=40&md5=656ab310cedf052101c74fe228c5afea

DOI: 10.1038/s41598-021-82881-2

Abstract:
Acute hepatopancreatic necrosis disease (AHPND) caused by PirABVP-producing strain of Vibrio parahaemolyticus, VPAHPND, has seriously impacted the shrimp production. Although the VPAHPND toxin is known as the VPAHPND virulence factor, a receptor that mediates its action has not been identified. An in-house transcriptome of Litopenaeus vannamei hemocytes allows us to identify two proteins from the aminopeptidase N family, LvAPN1 and LvAPN2, the proteins of which in insect are known to be receptors for Cry toxin. The membrane-bound APN, LvAPN1, was characterized to determine if it was a VPAHPND toxin receptor. The increased expression of LvAPN1 was found in hemocytes, stomach, and hepatopancreas after the shrimp were challenged with either VPAHPND or the partially purified VPAHPND toxin. LvAPN1 knockdown reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VPAHPND bacteria in the stomach after VPAHPND toxin challenge. In addition, LvAPN1 silencing prevented the toxin from causing severe damage to the hemocytes and sustained both the total hemocyte count (THC) and the percentage of living hemocytes. We found that the rLvAPN1 directly bound to both rPirAVP and rPirBVP toxins, supporting the notion that silencing of LvAPN1 prevented the VPAHPND toxin from passing through the cell membrane of hemocytes. We concluded that the LvAPN1 was involved in AHPND pathogenesis and acted as a VPAHPND toxin receptor mediating the toxin penetration into hemocytes. Besides, this was the first report on the toxic effect of VPAHPND toxin on hemocytes other than the known target tissues, hepatopancreas and stomach. Copyright: © 2021 Luangtrakul et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85104275795&doi=10.1371%2fJOURNAL.PPAT.1009463&partnerID=40&md5=3f0523b3daf9e6dca83822b3bf07f66b

DOI: 10.1371/JOURNAL.PPAT.1009463

Abstract:
While Vibrio parahaemolyticus (VPAHPND) has been identified as the cause of early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp, mechanisms of host response remain unknown. Understanding these processes is important to improve farming practices because this understanding will help to develop methods to enhance shrimp immunity. Pre-treatment of shrimp with 5-minute chronic non-lethal heat stress (NLHS) for 7 days was found to significantly increase Litopenaeus vannamei survival against VPAHPND infection. To elucidate the mechanism involved, mRNA and miRNA expression profiles from the hemocyte of L. vannamei challenged with VPAHPND after NLHS with corresponding control conditions were determined by RNA-Seq. A total of 2,664 mRNAs and 41 miRNAs were differentially expressed after the NLHS treatment and VPAHPND challenge. A miRNA-mRNA regulatory network of differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) was subsequently constructed and the interactions of DEMs in regulating the NLHS-induced immune-related pathways were identified. Transcriptomic data revealed that miRNA and mRNA interactions contribute to the modulation of NLHS-induced immune responses, such as the prophenoloxidase-activating system, hemocyte homeostasis, and antimicrobial peptide production, and these responses enhance VPAHPND resistance in L. vannamei. © 2020, The Author(s).

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Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85078202253&doi=10.1038%2fs41598-019-57409-4&partnerID=40&md5=8ccfabcfe731527e265e61834cae204b

DOI: 10.1038/s41598-019-57409-4

Abstract:
Viruses subvert macromolecular pathways in infected host cells to aid in viral gene amplification or to counteract innate immune responses. Roles for host-encoded, noncoding RNAs, including microRNAs, have been found to provide pro- and anti-viral functions. Recently, circular RNAs (circRNAs), that are generated by a nuclear back-splicing mechanism of pre-mRNAs, have been implicated to have roles in DNA virus-infected cells. This study examines the circular RNA landscape in uninfected and hepatitis C virus (HCV)infected liver cells. Results showed that the abundances of distinct classes of circRNAs were up-regulated or down-regulated in infected cells. Identified circRNAs displayed proviral effects. One particular up-regulated circRNA, circPSD3, displayed a very pronounced effect on viral RNA abundances in both hepatitis C virus- and Dengue virus-infected cells. Though circPSD3 has been shown to bind factor eIF4A3 that modulates the cellular nonsense-mediated decay (NMD) pathway, circPSD3 regulates RNA amplification in a pro-viral manner at a post-translational step, while eIF4A3 exhibits the anti-viral property of the NMD pathway. Findings from the global analyses of the circular RNA landscape argue that pro-, and likely, anti-viral functions are executed by circRNAs that modulate viral gene expression as well as host pathways. Because of their long half-lives, circRNAs likely play hitherto unknown, important roles in viral pathogenesis. © 2020 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85089768354&doi=10.1371%2fJOURNAL.PPAT.1008346&partnerID=40&md5=1fbb5f60bcf9ef4dfd7b586b8bd25aeb

DOI: 10.1371/JOURNAL.PPAT.1008346

Abstract:
While toxin-harboring Vibrio parahaemolyticus has been previously established as the causative agent of early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp, information on the mechanistic processes that happen in the host during infection is still lacking. Here, we examined the expression responses of the shrimp hemocyte transcriptome to V. parahaemolyticus AHPND (VPAHPND) by RNA sequencing (RNA-seq). Using libraries (SRA accession number SRP137285) prepared from shrimp hemocytes under experimental conditions, a reference library was de novo assembled for gene expression analysis of VPAHPND-challenged samples at 0, 3/6, and 48 h post infection (hpi). Using the library from 0-hpi as the control, 359 transcripts were found to be differentially expressed in the 3/6-hpi library, while 429 were differentially expressed in the 48-hpi library. The expression patterns reported in the RNA-seq of 9 representative genes such as anti-lipopolysaccharide factor (LvALF), crustin p (CRU), serpin 3 (SER), C-type lectin 3 (CTL), clottable protein 2 (CLO), mitogen-activated protein kinase kinase 4 (MKK4), P38 mitogen-activated protein kinase (P38), protein kinase A regulatory subunit 1 (PKA) and DNAJ homolog subfamily C member 1-like (DNJ) were validated by qRT-PCR. The expression of these genes was also analyzed in shrimp that were injected with the partially purified VPAHPND toxin. A VPAHPND toxin-responsive gene, LvALF was identified, and its function was characterized by RNA interference. LvALF knockdown resulted in significantly rapid increase of shrimp mortality caused by toxin injection. Protein-protein interaction analysis by molecular docking suggested that LvALF possibly neutralizes VPAHPND toxin through its LPS-binding domain. The data generated in this study provide preliminary insights into the differences in the immune response of shrimp to the bacterial and toxic aspect of VPAHPND as a disease. © 2018 Elsevier Ltd

Keyword: Early mortality syndrome; Hemocyte; Immune system; Shrimp; Transcriptome

Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85049894907&doi=10.1016%2fj.fsi.2018.06.054&partnerID=40&md5=55a37f0e31662475b6f9f499a86e9c03

DOI: 10.1016/j.fsi.2018.06.054

Abstract:
Acute hepatopancreatic necrosis disease, AHPND, caused by a specific strain of Vibrio parahaemolyticus (VPAHPND), results in great loss of global shrimp production. Despite this, studies on shrimp defense mechanisms protecting against AHPND are few. In this study, suppression subtractive hybridization (SSH) was performed to identify differentially expressed genes from white shrimp Litopenaeus vannamei hepatopancreas upon VPAHPND infection at the early stages: 3 and 6 h post challenge and in the late stage at 48 h post challenge. Hemocyanin (HMC) is the most abundant gene identified as the up-regulated gene in the SSH library. Various hemocyanin subunits such as hemocyanin (HMC), hemocyanin subunit L1 (HMCL1), L2 (HMCL2), L3 (HMCL3), and L4 (HMCL4) were analyzed for their expression levels upon VPAHPND infection and in response to challenge with partially purified toxin of VPAHPND by qRT-PCR. Only HMC was highly up-regulated at 3 and 6 h post challenge in response to VPAHPND challenge. Two HMC subunits, HMCL3 and HMCL4, were up-regulated in the early phase of VPAHPND toxin injection. Furthermore, all subunits were down-regulated in the late phase of VPAHPND and toxin challenges. The native hemocyanin protein purified from shrimp hemolymph, identified as mixture of HMC and HMCL1, exhibited agglutination activity on VPAHPND. Injecting the purified native hemocyanin along with VPAHPND into shrimp decreased the number of bacteria in the hemolymph as compared to the VPAHPND challenged control. Moreover, pre-incubation of the purified native hemocyanin and VPAHPND toxin prior to injection into shrimp resulted in the decrease of cumulative mortality of shrimp when compared to the control. In addition, protein-protein interaction analysis carried out by ELISA technique indicated that hemocyanin exhibited VPAHPND toxin-neutralizing activity through direct interaction with PirA subunit with a dissociation constant of 6.83 × 10−6 M. Our results indicated that upon VPAHPND infection the expression of hemocyanin was induced and hemocyanin functions might involve agglutination of invading VPAHPND and also neutralization of VPAHPND secreted toxin via direct interacting with the PirA protein. © 2018 Elsevier Ltd

Keyword: Hemocyanin; Shrimp immunity; Toxin neutralization; Vibrio parahaemolyticus AHPND

Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85044737812&doi=10.1016%2fj.dci.2018.03.010&partnerID=40&md5=501292ea143d83b08ecbb9d19765cc82

DOI: 10.1016/j.dci.2018.03.010

Abstract:
The anti-lipopolysaccharide factor isoform 3 (ALFPm3), the antimicrobial peptide from Penaeus monodon, possesses antibacterial and antiviral activities. Although the mechanism of action of ALFPm3 against bacteria has been revealed but its antiviral mechanism is still unclear. To further study how the ALFPm3 exhibits antiviral activity against the enveloped virus, white spot syndrome virus (WSSV), the ALFPm3-interacting proteins from WSSV were sought and identified five ALFPm3-interacting proteins, WSSV186, WSSV189, WSSV395, WSSV458, and WSSV471. Only the interaction between ALFPm3 and WSSV189, however, has been confirmed to be involved in anti-WSSV activity of ALFPm3. Herein, the interactions between ALFPm3 and rWSSV186, rWSSV395, rWSSV458, or rWSSV471 were further analyzed and confirmed by in vitro pull-down assay. Western blot analysis and immunoelectron microscopy showed that the uncharacterized proteins, WSSV186 and WSSV471, were nucleocapsid and envelope proteins, respectively. The decrease of shrimp survival after injection the shrimp with mixtures of each rWSSV protein, rALFPm3 and WSSV as compared to those injected with rALFPm3-neutralizing WSSV was clearly observed indicating that all rWSSV proteins could interfere with the neutralization effect of rALFPm3 on WSSV similar to that reported previously for WSSV189. Morphological change on WSSV after incubation with rALFPm3 was observed by TEM. The lysed WSSV virions were clearly observed where both viral envelope and nucleocapsid were dismantled. The results lead to the conclusion that the ALFPm3 displays direct effect on the viral structural proteins resulting in destabilization and breaking up of WSSV virions. © 2016 Elsevier Ltd

Keyword: ALFPm3; Antiviral activity; Host-viral interaction; Penaeus monodon; WSSV

Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85006802852&doi=10.1016%2fj.dci.2016.11.023&partnerID=40&md5=f7fe48652dc6c47aa5bbfa27af587a45

DOI: 10.1016/j.dci.2016.11.023