RESUMEN
As cellular chaperones, heat shock protein can facilitate viral infection in different steps of infection process. Previously, we have shown that the suppression of Litopenaeus vannamei (Lv)HSP90 not only results in a decline of white spot syndrome virus (WSSV) infection but also induces apoptosis in shrimp hemocyte cells. However, the mechanism underlying how LvHSP90 involved in WSSV infection remains largely unknown. In this study, a yeast two-hybrid assay and co-immunoprecipitation revealed that LvHSP90 interacts with the viral protein WSSV322 which function as an anti-apoptosis protein. Recombinant protein (r) LvHSP90 and rWSSV322 inhibited cycloheximide-induced hemocyte cell apoptosis in vitro. Co-silencing of LvHSP90 and WSSV322 in WSSV-infected shrimp led to a decrease in expression level of viral replication marker genes (VP28, ie-1) and WSSV copy number, while caspase 3/7 activity was noticeably induced. The number of apoptotic cells, confirmed by Hoechst 33342 staining assay and annexin V/PI staining, was significantly higher in LvHSP90 and WSSV322 co-silenced-shrimp than the control groups. Moreover, the co-silencing of LvHSP90 and WSSV322 triggered apoptosis by the mitochondrial pathway, resulting in the upregulation of pro-apoptotic protein expression (bax) and the downregulation of anti-apoptotic protein expression (bcl, Akt). This process also involved the release of cytochrome c (CytC) from the mitochondria and a decrease in mitochondrial membrane potential (MMP). These findings suggest that LvHSP90 interacts with WSSV322 to facilitate viral replication by inhibiting host apoptosis during WSSV infection.
Asunto(s)
Apoptosis , Proteínas de Artrópodos , Proteínas HSP90 de Choque Térmico , Hemocitos , Penaeidae , Virus del Síndrome de la Mancha Blanca 1 , Animales , Virus del Síndrome de la Mancha Blanca 1/fisiología , Penaeidae/inmunología , Penaeidae/virología , Penaeidae/genética , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Hemocitos/inmunología , Hemocitos/virología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The cellular endosomal sorting complex required for transport (ESCRT) system comprises five distinct components and is involved in many different physiological processes. Recent studies have shown that different viruses rely upon the host ESCRT system for viral infection. However, whether this system is involved in white spot syndrome virus (WSSV) infection remains unclear. Here, we identified 24 homologs of ESCRT subunits in kuruma shrimp, Marsupenaeus japonicus, and found that some key components were strongly upregulated in shrimp after WSSV infection. Knockdown of key components of the ESCRT system using RNA interference inhibited virus replication, suggesting that the ESCRT system is beneficial for WSSV infection. We further focused on TSG101, a crucial member of the ESCRT-I family that plays a central role in recognizing cargo and activating the ESCRT-II and ESCRT-III complexes. TSG101 colocalized with WSSV in hemocytes. The addition of N16 (a TSG101 inhibitor) markedly decreased WSSV replication. TSG101 and ALIX of the ESCRT system interact with WSSV envelope proteins. The host proteins TSG101, RAB5, and RAB7, the viral protein VP28, and DNA were detected in endosomes isolated from hemocytes of WSSV-infected shrimp. Knockdown of Rab5 and Rab7 expression reduced viral replication. Taken together, these results suggest that the ESCRT system is hijacked by WSSV for transport through the early to late endosome pathway. Our work identified a novel requirement for the intracellular trafficking and infection of WSSV, and provided novel therapeutic targets for the prevention and control of WSSV in shrimp aquaculture. IMPORTANCE: Viruses utilize the ESCRT machinery in a variety of strategies for their replication and infection. This study revealed that the interaction of ESCRT complexes with WSSV envelope proteins plays a crucial role in WSSV infection in shrimp. The ESCRT system is conserved in the shrimp Marsupenaeus japonicus, and 24 homologs of the ESCRT system were identified in the shrimp. WSSV exploits the ESCRT system for transport and propagation via the interaction of envelope proteins with host TSG101 and ALIX in an endosome pathway-dependent manner. Understanding the underlying mechanisms of WSSV infection is important for disease control and breeding in shrimp aquaculture.
Asunto(s)
Proteínas de Unión al ADN , Complejos de Clasificación Endosomal Requeridos para el Transporte , Penaeidae , Replicación Viral , Virus del Síndrome de la Mancha Blanca 1 , Animales , Virus del Síndrome de la Mancha Blanca 1/fisiología , Virus del Síndrome de la Mancha Blanca 1/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Penaeidae/virología , Penaeidae/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Endosomas/metabolismo , Endosomas/virología , Hemocitos/virología , Hemocitos/metabolismo , Interacciones Huésped-Patógeno , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/genética , Interferencia de ARNRESUMEN
Shrimp hemocytes are the vital immune cells participating in innate immune response to defend against viruses. However, the lack of specific molecular markers for shrimp hemocyte hindered the insightful understanding of their functional clusters and differential roles in combating microbial infections. In this study, we used single-cell RNA sequencing to map the transcriptomic landscape of hemocytes from the white spot syndrome virus (WSSV)-infected Litopenaeus vannamei and conjointly analyzed with our previous published single-cell RNA sequencing technology data from the healthy hemocytes. A total of 16 transcriptionally distinct cell clusters were identified, which occupied different proportions in healthy and WSSV-infected hemocytes and exerted differential roles in antiviral immune response. Following mapping of the sequencing data to the WSSV genome, we found that all types of hemocytes could be invaded by WSSV virions, especially the cluster 8, which showed the highest transcriptional levels of WSSV genes and exhibited a cell type-specific antiviral response to the viral infection. Further evaluation of the cell clusters revealed the delicate dynamic balance between hemocyte immune response and viral infestation. Unsupervised pseudo-time analysis of hemocytes showed that the hemocytes in immune-resting state could be significantly activated upon WSSV infection and then functionally differentiated to different hemocyte subsets. Collectively, our results revealed the differential responses of shrimp hemocytes and the process of immune-functional differentiation post-WSSV infection, providing essential resource for the systematic insight into the synergistic immune response mechanism against viral infection among hemocyte subtypes. IMPORTANCE: Current knowledge of shrimp hemocyte classification mainly comes from morphology, which hinder in-depth characterization of cell lineage development, functional differentiation, and different immune response of hemocyte types during pathogenic infections. Here, single-cell RNA sequencing was used for mapping hemocytes during white spot syndrome virus (WSSV) infection in Litopenaeus vannamei, identifying 16 cell clusters and evaluating their potential antiviral functional characteristics. We have described the dynamic balance between viral infestation and hemocyte immunity. And the functional differentiation of hemocytes under WSSV stimulation was further characterized. Our results provided a comprehensive transcriptional landscape and revealed the heterogeneous immune response in shrimp hemocytes during WSSV infection.
Asunto(s)
Proteínas de Artrópodos , Hemocitos , Interacciones Microbiota-Huesped , Penaeidae , RNA-Seq , Análisis de Expresión Génica de una Sola Célula , Virus del Síndrome de la Mancha Blanca 1 , Animales , Proteínas de Artrópodos/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Regulación de la Expresión Génica , Hemocitos/citología , Hemocitos/inmunología , Hemocitos/metabolismo , Hemocitos/virología , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/inmunología , Penaeidae/citología , Penaeidae/genética , Penaeidae/inmunología , Penaeidae/virología , Virus del Síndrome de la Mancha Blanca 1/genética , Virus del Síndrome de la Mancha Blanca 1/inmunologíaRESUMEN
Polydnaviruses are dsDNA viruses associated with endoparasitoid wasps. Delivery of the virus during parasitization of a caterpillar and subsequent virus gene expression is required for production of an amenable environment for parasitoid offspring development. Consequently, understanding of Polydnavirus gene function provides insight into mechanisms of host susceptibility and parasitoid wasp host range. Polydnavirus genes predominantly are arranged in multimember gene families, one of which is the vinnexins, which are virus homologues of insect gap junction genes, the innexins. Previous studies of Campoletis sonorensis Ichnovirus Vinnexins using various heterologous systems have suggested the four encoded members may provide different functionality in the infected caterpillar host. Here, we expressed two of the members, vnxG and vnxQ2, using recombinant baculoviruses in susceptible host, the caterpillar Heliothis virescens. Following intrahemocoelic injections, we observed that >90% of hemocytes (blood cells) were infected, producing recombinant protein. Larvae infected with a vinnexin-recombinant baculovirus exhibited significantly reduced molting rates relative to larvae infected with a control recombinant baculovirus and mock-infected larvae. Similarly, larvae infected with vinnexin-recombinant baculoviruses were less likely to survive relative to controls and showed reduced ability to encapsulate chromatography beads in an immune assay. In most assays, the VnxG protein was associated with more severe pathology than VnxQ2. Our findings support a role for Vinnexins in CsIV and more broadly Ichnovirus pathology in infected lepidopteran hosts, particularly in disrupting multicellular developmental and immune physiology.
Asunto(s)
Baculoviridae/genética , Interacciones Microbiota-Huesped , Larva/crecimiento & desarrollo , Mariposas Nocturnas/virología , Polydnaviridae/genética , Proteínas Virales/genética , Animales , Encapsulación Celular , Hemocitos/virología , Larva/virología , Polydnaviridae/metabolismo , Proteínas RecombinantesRESUMEN
Aedes aegypti mosquitoes are vectors for arboviruses of medical importance such as dengue (DENV) and Zika (ZIKV) viruses. Different innate immune pathways contribute to the control of arboviruses in the mosquito vector including RNA interference, Toll and Jak-STAT pathways. However, the role of cellular responses mediated by circulating macrophage-like cells known as hemocytes remains unclear. Here we show that hemocytes are recruited to the midgut of Ae. aegypti mosquitoes in response to DENV or ZIKV. Blockade of the phagocytic function of hemocytes using latex beads induced increased accumulation of hemocytes in the midgut and a reduction in virus infection levels in this organ. In contrast, inhibition of phagocytosis by hemocytes led to increased systemic dissemination and replication of DENV and ZIKV. Hence, our work reveals a dual role for hemocytes in Ae. aegypti mosquitoes, whereby phagocytosis is not required to control viral infection in the midgut but is essential to restrict systemic dissemination. Further understanding of the mechanism behind this duality could help the design of vector-based strategies to prevent transmission of arboviruses.
Asunto(s)
Aedes/citología , Aedes/virología , Virus del Dengue/fisiología , Hemocitos/inmunología , Hemocitos/virología , Virus Zika/fisiología , Aedes/anatomía & histología , Animales , Femenino , Hemocitos/fisiología , Mosquitos Vectores , Fagocitos/virología , FagocitosisRESUMEN
A wide range of hemocyte types exist in insects but a full definition of the different subclasses is not yet established. The current knowledge of the classification of silkworm hemocytes mainly comes from morphology rather than specific markers, so our understanding of the detailed classification, hemocyte lineage and functions of silkworm hemocytes is very incomplete. Bombyx mori nucleopolyhedrovirus (BmNPV) is a representative member of the baculoviruses and a major pathogen that specifically infects silkworms (Bombyx mori) and causes serious losses in sericulture industry. Here, we performed single-cell RNA sequencing (scRNA-seq) of hemocytes in BmNPV and mock-infected larvae to comprehensively identify silkworm hemocyte subsets and determined specific molecular and cellular characteristics in each hemocyte subset before and after viral infectmadion. A total of 20 cell clusters and their potential marker genes were identified in silkworm hemocytes. All of the hemocyte clusters were infected by BmNPV at 3 days after inoculation. Interestingly, BmNPV infection can cause great changes in the distribution of hemocyte types. The cells appearing in the infection group mainly belong to prohemocytes (PR), while plasmatocytes (PL) and granulocytes (GR) are very much reduced. Furthermore, we found that BmNPV infection suppresses the RNA interference (RNAi) and immune response in the major hemocyte types. In summary, our results revealed the diversity of silkworm hemocytes and provided a rich resource of gene expression profiles for a systems-level understanding of their functions in the uninfected condition and as a response to BmNPV.
Asunto(s)
Bombyx , Hemocitos , Nucleopoliedrovirus/inmunología , RNA-Seq , Análisis de la Célula Individual , Animales , Bombyx/inmunología , Bombyx/virología , Hemocitos/inmunología , Hemocitos/virología , Larva/inmunología , Larva/virologíaRESUMEN
Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) is a baculovirus that causes systemic infections in many arthropod pests. The specific molecular processes underlying the biocidal activity of AcMNPV on its insect hosts are largely unknown. We describe the transcriptional responses in two major pests, Spodoptera frugiperda (fall armyworm) and Trichoplusia ni (cabbage looper), to determine the host-pathogen responses during systemic infection, concurrently with the viral response to the host. We assembled species-specific transcriptomes of the hemolymph to identify host transcriptional responses during systemic infection and assessed the viral transcript abundance in infected hemolymph from both species. We found transcriptional suppression of chitin metabolism and tracheal development in infected hosts. Synergistic transcriptional support was observed to suggest suppression of immune responses and induction of oxidative stress indicating disease progression in the host. The entire AcMNPV core genome was expressed in the infected host hemolymph with a proportional high abundance detected for viral transcripts associated with replication, structure, and movement. Interestingly, several of the host genes that were targeted by AcMNPV as revealed by our study are also targets of chemical insecticides currently used commercially to control arthropod pests. Our results reveal an extensive overlap between biological processes represented by transcriptional responses in both hosts, as well as convergence on highly abundant viral genes expressed in the two hosts, providing an overview of the host-pathogen transcriptomic landscape during systemic infection.
Asunto(s)
Interacciones Huésped-Patógeno/genética , Proteínas de Insectos/genética , Mariposas Nocturnas/genética , Mariposas Nocturnas/virología , Nucleopoliedrovirus/fisiología , Agricultura , Animales , Quitina/genética , Quitina/metabolismo , Perfilación de la Expresión Génica , Genoma Viral , Hemocitos/inmunología , Hemocitos/virología , Hemolinfa/fisiología , Hemolinfa/virología , Larva/virología , Metabolismo de los Lípidos/genética , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/patogenicidad , Estrés Oxidativo/genética , Spodoptera/genética , Spodoptera/virología , Replicación ViralRESUMEN
As a downstream interactor of ß-catenin, Pangolin which is the homologous protein of the T cell factor/lymphoid enhancer factor (TCF/LEF) in vertebrates is less understood in the research field of immunity. In this study, two isoforms of Litopenaeus vannamei Pangolin (LvPangolin1 and LvPangolin2) were identified. Phylogenetic tree analysis revealed that all of the Pangolin proteins from invertebrates were represented the same lineage. The mRNA expression profiles of the LvPangolin1 and LvPangolin2 genes differed across different tissues. The expression of LvPangolin1 and the amount of LvPangolin1and LvPangolin2 combined (LvPangolinComb) were significantly increased in the haemocyte, intestine and gill but reduced in the hepatopancreas after white spot syndrome virus (WSSV) challenge. The inhibition of LvPangolin1 but not LvPangolinComb significantly reduced the survival rates of L. vannamei after WSSV infection, while significantly higher WSSV viral loads in both LvPangolin1-inhibited and LvPangolinComb-inhibited L. vannamei were observed. Knockdown of LvPangolin by RNAi could distinctly decrease the expression of antimicrobial peptide (AMP) genes and their related transcription factors. All of these results indicate that LvPangolin plays a positive role in the response to WSSV infection and that this may be mediated through regulating the immune signalling pathways which control the expression of AMPs with antiviral abilities.
Asunto(s)
Proteínas de Artrópodos/inmunología , Inmunidad Innata/inmunología , Penaeidae/inmunología , Factores de Transcripción TCF/inmunología , Virus del Síndrome de la Mancha Blanca 1/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/genética , Secuencia de Bases , Clonación Molecular , Hemocitos/inmunología , Hemocitos/metabolismo , Hemocitos/virología , Hepatopáncreas/inmunología , Hepatopáncreas/metabolismo , Hepatopáncreas/virología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/genética , Penaeidae/genética , Penaeidae/virología , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Análisis de Secuencia de ADN , Análisis de Supervivencia , Factores de Transcripción TCF/clasificación , Factores de Transcripción TCF/genética , Transcriptoma/inmunología , Virus del Síndrome de la Mancha Blanca 1/fisiologíaRESUMEN
Echinoderms are important marine organisms that live in a wide range from the intertidal zone to the abyssal zone. Members of this phylum are prone to dramatic population fluctuations that may trigger dramatic shifts in ecosystem structure. Despite the extremely complex nature of the marine environment, the immune systems of echinoderms induce a complex innate immune response to prokaryotic and eukaryotic pathogens. Previous studies showed that many echinoderm disease outbreaks were associated with specific bacteria, whereas recent scientific investigations using newly developed technologies revealed the amazing diversity of viruses in seawater. Viruses are potential pathogens of several infectious diseases of marine echinoderms. We reviewed the discovery of viruses in echinoderms and discussed the relationship between viruses and diseases for the first time. We further summarized the research progress of the potential immune-related genes and signal pathways induced by viruses and poly (I:C). Additionally, numbers of studies showed that active substances extracted from echinoderms, or the compounds synthesized from these substances, have significant antihuman virus ability. This result suggests that the active substances derived from echinoderms provide potential antiviral protection for the organism, which may provide future research directions for the antiviral immunity of echinoderms. Thus, this review also collected information on the antiviral activities of biologically active substances from echinoderms, which may pave the way for new trends in antiviral immunity for echinoderms and antiviral drugs in humans.
Asunto(s)
Factores de Restricción Antivirales/inmunología , Organismos Acuáticos/inmunología , Equinodermos/inmunología , Inmunidad Innata/inmunología , Virus/inmunología , Animales , Péptidos Antimicrobianos/inmunología , Péptidos Antimicrobianos/metabolismo , Factores de Restricción Antivirales/genética , Organismos Acuáticos/genética , Organismos Acuáticos/virología , Equinodermos/genética , Equinodermos/virología , Ecosistema , Hemocitos/inmunología , Hemocitos/metabolismo , Hemocitos/virología , Humanos , Inmunidad Humoral/inmunología , Inmunidad Innata/genética , Fagocitosis/inmunología , Virus/crecimiento & desarrollo , Virus/aislamiento & purificaciónRESUMEN
The apoptosis of hemocytes plays an essential function in shrimp immune defense against pathogen invasions. In order to further elucidate the differential apoptotic responses of the granulocytes and the hyalinocytes in Fenneropenaeus chinensis post WSSV infection, the characteristics of apoptotic dynamics and viral proliferation in total hemocytes and hemocyte subpopulations were respectively investigated in the present work. The results showed that the apoptotic rate of hemocytes changed significantly, and the apoptosis-related genes also showed significantly differential expression responses during WSSV infection. Interestingly, we found that the apoptotic rate of virus-negative hemocytes was significantly higher than that of virus-positive hemocytes in the early stage of WSSV infection, while it was significantly lower than that of virus-positive cells in the middle and late infection stages. The difference of apoptosis between virus-positive and virus-negative hemocytes seems to be an important way for the WSSV to destroy the host's immune system and facilitate the virus spread at different infection stages. It was further found that the apoptosis rate of granulocytes was always significantly higher than that of hyalinocytes during WSSV infection, indicating that granulocytes have a stronger apoptotic response to WSSV infection. Moreover, a higher viral load was detected in granulocytes, and the density of granulocytes decreased more rapidly post WSSV infection, indicating that the granulocytes are more susceptible and vulnerable to WSSV infection compared with the hyalinocytes. These results collectively demonstrated that the apoptotic response in shrimp hemocytes was significantly influenced by the WSSV infection, and the differential apoptotic response of granulocytes and hyalinocytes to WSSV indicated the differences of antiviral mechanisms between the two hemocyte subpopulations.
Asunto(s)
Enfermedades de los Animales/virología , Apoptosis , Hemocitos/metabolismo , Hemocitos/virología , Interacciones Huésped-Patógeno , Penaeidae/virología , Virus del Síndrome de la Mancha Blanca 1/fisiología , Animales , Apoptosis/genética , Apoptosis/inmunología , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Granulocitos/inmunología , Granulocitos/metabolismo , Hemocitos/inmunología , Interacciones Huésped-Patógeno/inmunologíaRESUMEN
Double-stranded RNA (dsRNA) is employed to down-regulate the expression of specific genes of shrimp viral pathogens through the RNA interference (RNAi) pathway. The administration of dsRNA into shrimp has been shown to be an effective strategy to block yellow head virus (YHV) progression. In this study, a vector (pLVX-AcGFP1-N1) was developed to introduce a long-hairpin RNA (lhRNA) silencing cassette under a CMV promoter, so-called "pLVX-lhRdRp", against the RNA-dependent RNA polymerase (RdRp) gene of YHV. A primary culture of hemocytes isolated from Penaeus monodon was transfected with the pLVX-lhRdRp vector, generating transcripts of lhRNAs as early as 12 h post transfection. Twelve hours prior to YHV challenge, the primary hemocyte cell culture was transfected with pLVX-lhRdRp, whereas control groups were transfected with pLVX-AcGFP1-N1 or no transfection. The group treated with pLVX-lhRdRp significantly suppressed YHV replication at 24-72 h after YHV challenge. The results from RT-PCR and immunohistochemistry confirmed that both mRNA and protein expression of YHV were effectively inhibited by the pLVX-lhRdRp vector. Thus, our hemocyte culture and dsRNA expression plasmid with constitutive promoter have potential as a platform to test DNA constructs expressing long-hairpin RNA against pathogenic viral infection and as a RNAi-based DNA vaccine in shrimp.
Asunto(s)
Hemocitos/virología , Penaeidae/virología , Interferencia de ARN , ARN Bicatenario/metabolismo , Roniviridae/fisiología , Replicación Viral , AnimalesRESUMEN
Emerging studies have indicated that some penaeidins restrict virus infection; however, the mechanism(s) involved are poorly understood. In the present study, we uncovered that penaeidins are a novel family of antiviral effectors against white spot syndrome virus (WSSV), which antagonize the envelope proteins to block viral entry. We found that the expression levels of four identified penaeidins from Litopenaeus vannamei, including BigPEN, PEN2, PEN3, and PEN4, were significantly induced in hemocytes during the early stage of WSSV infection. Knockdown of each penaeidin in vivo via RNA interference resulted in elevated viral loads and rendered shrimp more susceptible to WSSV, while the survival rate was rescued via the injection of recombinant penaeidins. All penaeidins, except PEN4, were shown to interact with several envelope proteins of WSSV, and all four penaeidins were observed to be located on the outer surface of the WSSV virion. Co-incubation of each recombinant penaeidin with WSSV inhibited virion internalization into hemocytes. More importantly, we found that PEN2 competitively bound to the envelope protein VP24 to release it from polymeric immunoglobulin receptor (pIgR), the cellular receptor required for WSSV infection. Moreover, we also demonstrated that BigPEN was able to bind to VP28 of WSSV, which disrupted the interaction between VP28 and Rab7 - the Rab GTPase that contributes to viral entry by binding with VP28. Taken together, our results demonstrated that penaeidins interact with the envelope proteins of WSSV to block multiple viral infection processes, thereby protecting the host against WSSV.
Asunto(s)
Antivirales/farmacología , Penaeidae/virología , Péptidos , Virus del Síndrome de la Mancha Blanca 1/efectos de los fármacos , Animales , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Expresión Génica , Hemocitos/virología , Péptidos/genética , Péptidos/farmacología , Proteínas del Envoltorio Viral/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
C-type lectins (CTLs) are key recognition proteins in shrimp immunity. A few years ago we reviewed sequence information, ligand specificity, expression profiles and specific functions of the shrimp CTLs. Since then, multiple integrated studies that implemented biochemical approaches using both the native and recombinant proteins, functional genetic approaches using RNA interference, and mechanistic studies by analyzing protein-protein interactions were carried out. Results from these rigorous studies revealed the functions and mechanisms of action of selected members of the shrimp CTL family. This review focuses on this new knowledge, that includes unique structural aspects, functions, and mechanisms in host-pathogen interactions, the functional relevance of regions other than the C-type lectin domain, and the regulation of transcription of shrimp CTLs. Thus, this review aims to provide a detailed update of recent studies that have contributed to our better understanding of the shrimp immune events that involve CTL functions.
Asunto(s)
Proteínas de Artrópodos/inmunología , Expresión Génica/inmunología , Lectinas Tipo C/inmunología , Penaeidae/inmunología , Virus del Síndrome de la Mancha Blanca 1/inmunología , Animales , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Endosomas/inmunología , Endosomas/metabolismo , Endosomas/virología , Hemocitos/inmunología , Hemocitos/metabolismo , Hemocitos/virología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/inmunología , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Penaeidae/genética , Penaeidae/virología , Virus del Síndrome de la Mancha Blanca 1/fisiologíaRESUMEN
microRNAs (miRNAs) are known to regulate various immune functions by silencing the target genes in both vertebrates and invertebrates. However, in mud crab Scylla paramamosain, the role of miRNAs during the response to virus invasion remains unclear. To investigate the roles of miRNAs in S. paramamosain during virus infection, the mud crab was challenged with white spot syndrome virus (WSSV) and then subjected to the transcriptional analysis at different conditions. The results of high-throughput sequencing revealed that 940,379 and 1,306,023 high-quality mappable reads were detected in the hemocyte of normal and WSSV-infected mud crabs, respectively. Besides, the total number of 261 unique miRNAs were identified. Among them, 131 miRNAs were specifically expressed in the hemocytes of normal mud crabs, 46 miRNAs were specifically transcribed in those of WSSV-infected individuals, the other 84 miRNAs were expressed in both normal and WSSV-infected individuals. Furthermore, a number of 152 (89 down-regulated and 63 up-regulated) miRNAs were found to be differentially expressed in the WSSV-infected hemocytes, normalized to the controls. The identified miRNAs were subjected to GO analysis and target gene prediction and the results suggested that the differentially regulated miRNAs were mainly correlated with the changes of the immune responses of the hemocytes, including phagocytosis, melanism, and apoptosis as well. Taken together, the results demonstrated that the expressed miRNAs during the virus infection were mainly involved in the regulation of immunological pathways in mud crabs. Our findings not only enrich the understanding of the functions of miRNAs in the innate immune system but also provide some novel potential targets for the prevention of WSSV infection in crustaceans.
Asunto(s)
Braquiuros/genética , Braquiuros/virología , Infecciones por Virus ADN/veterinaria , MicroARNs/genética , Animales , Braquiuros/inmunología , Biología Computacional , Infecciones por Virus ADN/inmunología , Perfilación de la Expresión Génica , Hemocitos/inmunología , Hemocitos/virología , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunidad Innata/genética , Fagocitosis , Virus del Síndrome de la Mancha Blanca 1RESUMEN
Litopenaeus vannamei, as an important marine aquaculture species, has attracted more and more attentions in past several years. More recently people got its genome fine mapping, which unveiled a gene treasure. In this study, we have identified a novel trypsin-like protein which came from previous WSSV-infected shrimp plasma iTRAQ data. This protein is a 39â¯kDa protein with 363 amino acids. It contains a conserved trypsin-domain and could be strongly induced with WSSV infection. Interestingly, knockdown of this protein made shrimps vulnerable to WSSV infection. Further exploration unveiled that this fragility was probably due to the fact that knockdown of this protein could cause shrimp hemocytes apoptosis, which indicated that this protein played key roles in preventing shrimp hemocytes from apoptosis. To further explore how LvTLAP protected shrimp hemocytes from apoptosis, GST pull down assay was applied to screen LvTLAP interacting protein in shrimp plasma. L. vannamei growth and transformation-dependent-like protein (LvGTD-like protein) was identified as a LvTLAP interacting protein, which played proapoptotic roles in cells. Thus, a possible explanation for LvTLAP anti-apoptosis activity was that this protein could block LvGTD-like protein proapoptotic activity to protect shrimp hemocytes from death. In general, our study has uncovered a novel WSSV responsive shrimp plasma protein, which played key roles in shrimp hemocytes anti-apoptosis and shrimp against WSSV infection.
Asunto(s)
Proteínas Sanguíneas/inmunología , Penaeidae/inmunología , Penaeidae/virología , Virus del Síndrome de la Mancha Blanca 1/patogenicidad , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Apoptosis/inmunología , Apoptosis/fisiología , Secuencia de Bases , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , ADN/genética , Técnicas de Silenciamiento del Gen , Hemocitos/metabolismo , Hemocitos/patología , Hemocitos/virología , Penaeidae/genéticaRESUMEN
Kuruma prawn, Marsupenaeus japonicus, has the third largest annual yield among shrimp species with vital economic significance in China. White spot syndrome virus (WSSV) is a great threat to the global shrimp farming industry and results in high mortality. Pellino, a highly conserved E3 ubiquitin ligase, has been found to be an important modulator of the Toll-like receptor (TLR) signaling pathways that participate in the innate immune response and ubiquitination. In the present study, the Pellino gene from Marsupenaeus japonicus was identified. A qRT-PCR assay showed the presence of MjPellino in all the tested tissues and revealed that the transcript level of this gene was significantly upregulated in both the gills and hemocytes after challenge with WSSV and Vibrio parahaemolyticus. The function of MjPellino was further verified at the protein level. The results of the three-dimensional modeling and protein-protein docking analyses and a GST pull-down assay revealed that the MjPellino protein was able to bind to the WSSV envelope protein VP26. In addition, the knockdown of MjPellino in vivo significantly decreased the expression of MjAMPs. These results suggest that MjPellino might play an important role in the immune response of kuruma prawn.
Asunto(s)
Proteínas de Artrópodos/genética , Penaeidae/genética , Ubiquitina-Proteína Ligasas/genética , Vibriosis/genética , Secuencia de Aminoácidos/genética , Animales , Proteínas de Artrópodos/aislamiento & purificación , China , Perfilación de la Expresión Génica/métodos , Hemocitos/microbiología , Hemocitos/virología , Humanos , Inmunidad Innata/genética , Penaeidae/microbiología , Penaeidae/virología , Receptores Toll-Like/genética , Activación Transcripcional/genética , Vibriosis/microbiología , Vibrio parahaemolyticus/patogenicidad , Virus del Síndrome de la Mancha Blanca 1/genética , Virus del Síndrome de la Mancha Blanca 1/patogenicidadRESUMEN
White spot syndrome (WSS) caused by white spot syndrome virus (WSSV) is a severe infectious disease in shrimp aquaculture. To find effective therapeutics to control WSSV, it is indispensable to understand the innate immune responses of shrimp to WSSV infection. Previous report demonstrated that the Litopenaeus vannamei heat shock protein 70 (LvHSP70) could induce shrimp innate immunity against bacterial infection. Herein, we further investigate the role of LvHSP70 in anti-WSSV infection. The temporal expression of LvHSP70 was significantly upregulated 2.5- and 1.5-fold at 6 and 24â¯h post systemic WSSV infection suggesting that the LvHSP70 was a WSSV responsive gene. The recombinant protein of LvHSP70 (rLvHSP70) was produced in an Escherichia coli system and its effect in protection against WSSV infection was investigated. Intramuscularly injection of juvenile shrimp with 1â¯nmol of rLvHSP70 could significantly prolong 50% mortality of WSSV-infected shrimp from 3 days to 5 days as compared to the control group injected with bovine serum albumin (BSA). Consistently, the injection of rLvHSP70 resulted in 24-fold, 20-fold and 100-fold decrease in the viral copy number after 6, 12 and 24â¯h post injection, respectively, compared to the control shrimp injected with BSA. Interestingly, it was found that the rLvHSP70 enhanced the expression of the key gene in the prophenoloxidase (proPO) activating system, LvproPO, but reduced the expression of Lvcaspase2 and LvIAP in WSSV-infected shrimp. These results suggested that the LvHSP70 is an important molecule involved in antiviral defense in shrimp presumably via modulating the proPO system and apoptosis.
Asunto(s)
Proteínas de Artrópodos/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Penaeidae/inmunología , Penaeidae/virología , Virus del Síndrome de la Mancha Blanca 1/fisiología , Animales , Apoptosis , Proteínas de Artrópodos/administración & dosificación , Proteínas de Artrópodos/genética , Catecol Oxidasa/genética , Resistencia a la Enfermedad/genética , Precursores Enzimáticos/genética , Regulación de la Expresión Génica/inmunología , Proteínas HSP70 de Choque Térmico/administración & dosificación , Proteínas HSP70 de Choque Térmico/genética , Hemocitos/inmunología , Hemocitos/virología , Inmunidad Innata/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/genética , Tasa de Supervivencia , Regulación hacia Arriba/genéticaRESUMEN
Shrimp hemocyte iridescent virus (SHIV), which was first identified in white leg shrimp (Litopenaeus vannamei) in China in 2014, can cause extensive shrimp mortality and major economic losses in the shrimp farming industry in China. In this study, a novel real-time isothermal recombinase polymerase amplification (RPA) assay was developed using a TwistAmp exo kit for SHIV detection. First, five primers and a probe were designed for the major capsid protein gene (GenBank: KY681039.1) according to the TwistDx manual; next, the optimal primers were selected by a comparison experiment. The primers and probe were specific for SHIV and did not react with shrimp white spot syndrome virus (WSSV), shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp enterocytozoon hepatopenaei (EHP), and macrobrachium rosenbergii nodavirus (MrNV) samples, as well as pathogens of acute hepatopancreatic necrosis disease (AHPND). The RPA assay reached a detection limit of 11 copies per reaction according to probit regression analysis. In addition, RPA assay detected the positive plasmid samples at concentration of 1000 copies/µL within 16.04⯱â¯0.72â¯minâ¯at a single low operation temperature (39⯰C). The results proved that the proposed RPA method was an accurate, sensitive, affordable, and rapid detection tool that can be suitably applied for the diagnosis of SHIV in field conditions and in resource-poor settings.
Asunto(s)
Hemocitos/virología , Iridovirus/aislamiento & purificación , Penaeidae/virología , Reacción en Cadena de la Polimerasa/métodos , Recombinasas/metabolismo , Animales , Cartilla de ADN/genética , Iridovirus/genética , Plásmidos/genética , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The white spot syndrome virus (WSSV) causes mass mortalities in the aquaculture of shrimps worldwide. The mud shrimp Austinogebia edulis (Ngoc-Ho & Chan, 1992) is an economically important sea food item occurring along the west coast of Taiwan. While the population of A. edulis began to decrease with some fluctuations in the last decade, the current study aims to discover the causes for such sporadic population decline. This study explores the effects of microbial pathogens and innate immunity on the populations of A. edulis. Here, we report firstly about WSSV infection of A. edulis from the coastal zone of western Taiwan which is one of the possible causes of population decrease of A. edulis in Shengang. However, WSSV infection is not the only reason for its population decrease because a similar infection rate of WSSV was found in Wangong. Population changes may be related to both environmental pollution stress and WSSV. Both factors likely caused a massive reduction of hemocytes and an abnormal increase of phenoloxidase and superoxide dismutase activity, which were spectrophotometrically measured. Since there is no effective way to treat WSSV infection, improving the coastal environment appears the most effective way to increase the population size of feral shrimps.