Molecular cloning and functional analyses of C-C motif chemokine ligand 3 (CCL3) in mandarin fish Siniperca chuatsi.

Chemokines are critical molecules involved in immune reaction and immune system homeostasis, and some chemokines play a role in antiviral immunity. It is not known if the C-C motif chemokine ligand 3 (CCL3), a member of the CC chemokine family, possesses antiviral properties in fish. In this study, a ccl3 was cloned from the mandarin fish (Siniperca chuatsi), and it has an open reading frame (ORF) of 276 base pairs, which are predicted to encode a 91-amino acid peptide. Mandarin fish CCL3 revealed conserved sequence features with four cysteine residues and closely relationships with the CCL3s from other vertebrates based on the sequence alignment and phylogenetic analysis. The transcripts of ccl3 were notably enriched in immune-related organs, such as spleen and gills in healthy mandarin fish, and the ccl3 was induced in the isolated mandarin fish brain (MFB) cells following infection with infectious spleen and kidney necrosis virus (ISKNV). Moreover, in MFB cells, overexpression of CCL3 induced immune factors, such as IL1ß, TNFα, MX, IRF1 and IFNh, and exhibited antiviral activity against ISKNV. This study sheds light on the immune role of CCL3 in immune response of mandarin fish, and its antiviral defense mechanism is of interest for further investigation.

Fish ELOVL7a is involved in virus replication via lipid metabolic reprogramming.

The elongation of very long chain fatty acids (ELOVL) proteins are key rate-limiting enzymes that catalyze fatty acid synthesis to form long chain fatty acids. ELOVLs also play regulatory roles in the lipid metabolic reprogramming induced by mammalian viruses. However, little is known about the roles of fish ELOVLs during virus infection. Here, a homolog of ELOVL7 was cloned from Epinephelus coioides (EcELOVL7a), and its roles in red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV) infection were investigated. The transcription level of EcELOVL7a was significantly increased upon RGNNV and SGIV infection or other pathogen-associated molecular patterns stimulation in grouper spleen (GS) cells. Subcellular localization analysis showed that EcELOVL7a encoded an endoplasmic reticulum (ER) related protein. Overexpression of EcELOVL7a promoted the viral production and virus release during SGIV and RGNNV infection. Furthermore, the lipidome profiling showed that EcELOVL7a overexpression reprogrammed cellular lipid components in vitro, evidenced by the increase of glycerophospholipids, sphingolipids and glycerides components. In addition, VLCFAs including FFA (20:2), FFA (20:4), FFA (22:4), FFA (22:5) and FFA (24:0), were enriched in EcELOVL7a overexpressed cells. Consistently, EcELOVL7a overexpression upregulated the transcription level of the key lipid metabolic enzymes, including fatty acid synthase (FASN), phospholipase A 2α (PLA 2α), and cyclooxygenases -2 (COX-2), LPIN1, and diacylglycerol acyltransferase 1α (DGAT1α). Together, our results firstly provided the evidence that fish ELOVL7a played an essential role in SGIV and RGNNV replication by reprogramming lipid metabolism.

Study of the antivirus function mediated by STING in Micropterus salmoides.

Stimulator of interferon genes (STING) has been demonstrated as a critical mediator in the innate immune response to cytosolic DNA and RNA derived from different pathogens. While the role of Micropterus salmoides STING (MsSTING) in largemouth bass virus is still unknown. In this study, RT-qPCR assay and Western-blot assay showed that the expression levels of MsSTING and its downstream genes were up-regulated after LMBV infection. Pull down experiment proved that a small peptide called Fusion peptide (FP) that previously reported to target to marine and human STING as a selective inhibitor also interacted with MsSTING in vitro. Comparing with the RNA-seq of Largemouth bass infected with LMBV singly, 326 genes were significantly up-regulated and 379 genes were significantly down-regulated in the FP plus LMBV group in which Largemouth bass was treatment with FP before LMBV-challenged. KEGG analysis indicated that the differentially expressed genes (DEGs) were mainly related to signaling transduction, infectious disease viral, immune system and endocrine system. Besides, the survival rate of LMBV-infected largemouth bass was highly decreased following FP treatment. Taken together, our study showed that MsSTING played an important role in immune response against LMBV infection.

Berberine inhibits SGIV replication by suppressing inflammatory response and oxidative stress.

Singapore grouper iridovirus (SGIV) is one of the major infectious diseases responsible for high mortality and huge economic losses in the grouper aquaculture industry. Berberine (BBR), a naturally occurring plant alkaloid, is a phytochemical having a variety of biological properties, such as antiviral, antioxidant, and anti-inflammatory effects. In this work, we used an in vitro model based on Western blot, ROS fluorescence probe, and real-time quantitative PCR (qRT-PCR) to examine the antiviral qualities of BBR against SGIV. The outcomes demonstrated that varying BBR concentrations could significantly inhibit the replication of SGIV. In addition, BBR greatly inhibited the production of genes associated with pro-inflammatory cytokines in SGIV-infected or SGIV-uninfected GS cells based on qRT-PCR data. Subsequent investigations demonstrated that BBR suppressed the expression of the promoter activity of NF-κB and NF-κB-p65 protein. Additionally, BBR reduced the phosphorylation of ERK 1/2, JNK, and p38. Furthermore, BBR also inhibits SGIV-induced ROS production by upregulating the expression of antioxidant-related genes. In conclusion, BBR is a viable therapy option for SGIV infection due to its antiviral properties.

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 CRL5-SPSB3 ubiquitin ligase targets nuclear cGAS for degradation.

Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis of 2'3'-cyclic GMP-AMP (cGAMP)1-7. The indiscriminate activity of cGAS towards DNA demands tight regulatory mechanisms that are necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA8-15. However, the fate of nuclear cGAS and its role in cell physiology remains unclear. Here we show that the ubiquitin proteasomal system (UPS) degrades nuclear cGAS in cycling cells. We identify SPSB3 as the cGAS-targeting substrate receptor that associates with the cullin-RING ubiquitin ligase 5 (CRL5) complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy structure of nucleosome-bound cGAS in a complex with SPSB3 reveals a highly conserved Asn-Asn (NN) minimal degron motif at the C terminus of cGAS that directs SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon signalling, conferring heightened protection against infection by DNA viruses. Our research defines protein degradation as a determinant of cGAS regulation in the nucleus and provides structural insights into an element of cGAS that is amenable to therapeutic exploitation.

Zebrafish TMEM47 is an effective blocker of IFN production during RNA and DNA virus infection.

IMPORTANCE: Mitochondrial antiviral signaling protein (MAVS) and stimulator of interferon (IFN) genes (STING) are key adaptor proteins required for innate immune responses to RNA and DNA virus infection. Here, we show that zebrafish transmembrane protein 47 (TMEM47) plays a critical role in regulating MAVS- and STING-triggered IFN production in a negative feedback manner. TMEM47 interacted with MAVS and STING for autophagic degradation, and ATG5 was essential for this process. These findings suggest the inhibitory function of TMEM47 on MAVS- and STING-mediated signaling responses during RNA and DNA virus infection.

Opposing effects of deubiquitinase OTUD3 in innate immunity against RNA and DNA viruses.

Retinoic acid-inducible-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and cyclic GMP-AMP synthase (cGAS) genes encode essential cytosolic receptors mediating antiviral immunity against viruses. Here, we show that OTUD3 has opposing role in response to RNA and DNA virus infection by removing distinct types of RIG-I/MDA5 and cGAS polyubiquitination. OTUD3 binds to RIG-I and MDA5 and removes K63-linked ubiquitination. This serves to reduce the binding of RIG-I and MDA5 to viral RNA and the downstream adaptor MAVS, leading to the suppression of the RNA virus-triggered innate antiviral responses. Meanwhile, OTUD3 associates with cGAS and targets at Lys279 to deubiquitinate K48-linked ubiquitination, resulting in the enhancement of cGAS protein stability and DNA-binding ability. As a result, Otud3-deficient mice and zebrafish are more resistant to RNA virus infection but are more susceptible to DNA virus infection. These findings demonstrate that OTUD3 limits RNA virus-triggered innate immunity but promotes DNA virus-triggered innate immunity.

Endogenous Retroviruses Augment Amphibian (Xenopus laevis) Tadpole Antiviral Protection.

The global amphibian declines are compounded by infections with members of the Ranavirus genus such as Frog Virus 3 (FV3). Premetamorphic anuran amphibians are believed to be significantly more susceptible to FV3 while this pathogen targets the kidneys of both pre- and postmetamorphic animals. Paradoxically, FV3-challenged Xenopus laevis tadpoles exhibit lower kidney viral loads than adult frogs. Presently, we demonstrate that X. laevis tadpoles are intrinsically more resistant to FV3 kidney infections than cohort-matched metamorphic and postmetamorphic froglets and that this resistance appears to be epigenetically conferred by endogenous retroviruses (ERVs). Using a X. laevis kidney-derived cell line, we show that enhancing ERV gene expression activates cellular double-stranded RNA-sensing pathways, resulting in elevated mRNA levels of antiviral interferon (IFN) cytokines and thus greater anti-FV3 protection. Finally, our results indicate that large esterase-positive myeloid-lineage cells, rather than renal cells, are responsible for the elevated ERV/IFN axis seen in the tadpole kidneys. This conclusion is supported by our observation that CRISPR-Cas9 ablation of colony-stimulating factor-3 results in abolished homing of these myeloid cells to tadpole kidneys, concurrent with significantly abolished tadpole kidney expression of both ERVs and IFNs. We believe that the manuscript marks an important step forward in understanding the mechanisms controlling amphibian antiviral defenses and thus susceptibility and resistance to pathogens like FV3. IMPORTANCE Global amphibian biodiversity is being challenged by pathogens like the Frog Virus 3 (FV3) ranavirus, underlining the need to gain a greater understanding of amphibian antiviral defenses. While it was previously believed that anuran (frog/toad) amphibian tadpoles are more susceptible to FV3, we demonstrated that tadpoles are in fact more resistant to this virus than metamorphic and postmetamorphic froglets. We showed that this resistance is conferred by large myeloid cells within the tadpole kidneys (central FV3 target), which possess an elevated expression of endogenous retroviruses (ERVs). In turn, these ERVs activate cellular double-stranded RNA-sensing pathways, resulting in a greater expression of antiviral interferon cytokines, thereby offering the observed anti-FV3 protection.

Grouper USP12 exerts antiviral activity against nodavirus infection.

The ubiquitin-specific proteases (USPs) have attracted particular attention due to their multiple functions in different biological processes. USP12, a member of the USP family, has been demonstrated to exert critical roles in diverse cellular processes, including cell death, cancer and antiviral immunity. Here, we cloned a USP12 homolog from orange spotted grouper (Epinephelus coioides, E. coioides), and its roles in fish RNA virus replication were investigated. EcUSP12 contained a 1119-bp open reading frame (ORF) encoding a 372-amino acid polypeptide, which shared 100.00% and 91.32% identity with USP12 homolog of Etheostoma cragini and Homo sapiens, respectively. Sequence analysis indicated that EcUSP12 contained a conserved peptidase-C19G domain (aa 40-369). qPCR analysis showed that EcUSP12 transcript was most abundant in head kidney and spleen of grouper E. coioides. The expression of EcUSP12 was significantly upregulated in grouper spleen (GS) cells in response to red-spotted grouper nervous necrosis virus (RGNNV) infection. Subcellular localization analysis showed that EcUSP12 was evenly distributed throughout the cytoplasm, and mainly co-localized with endoplasmic reticulum (ER). Interestingly, during RGNNV infection, the endogenous distribution of EcUSP12 was obviously altered, and mostly overlapped with viral coat protein (CP). Co-Immunoprecipitation (Co-IP) assay indicated that EcUSP12 interacted with viral CP. In addition, overexpression of EcUSP12 significantly inhibited the replication of RGNNV in vitro, as evidenced by the decrease in viral gene transcription and protein synthesis during infection. Consistently, knockdown of EcUSP12 by small interfering RNA (siRNA) promoted the replication of RGNNV. Furthermore, EcUSP12 overexpression also increased the transcription level of inflammatory factors and interferon-related genes, including tumor necrosis factor α (TNF-α), interleukin (IL)-1ß, IL-6, IL-8, interferon regulatory factor 3 (IRF3), and IRF7. Taken together, our results demonstrated that EcUSP12, as a positive regulator of IFN signaling, interacted with viral CP to inhibit virus infection.

Functional analysis of a novel MHC-Iα genotype in orange-spotted grouper: Effects on Singapore grouper iridovirus (SGIV) replication and apoptosis.

The classical major histocompatibility complex class I (MHC-Ⅰ) molecule plays a key role in vertebrate immune response for its important functions in antigen presentation and immune regulation. MHC pathway is closely related to many diseases involving autoimmunity, antigen intrusion and inflammation. However, rare literatures about the effect of MHC-I on fish cells apoptosis were reported. In this study, a novel type of MHC-Ⅰα genotype from orange-spotted grouper (named EcMHC-ⅠA*01) were cloned and characterized. It shared a 77% identity to its Epinephelus coioides MHC-Iα homology that has been uploaded to NCBI (ACZ97571.1). Molecular characterization analysis showed that EcMHC-ⅠA*01 encodes a 357-amino-acid protein, containing a signal peptide，α1，α2，α3, Cytoplasmic (Cyt) and Transmembrane (TM) domains. Tissue expression pattern showed that EcMHC-ⅠA*01 was extensively distributed in twelve selected tissues, with higher expression in the gill, intestine and skin. The expression of EcMHC-ⅠA*01 in grouper liver and spleen tissues were significantly induced by different stimuli (Zymosan A, LPS, Ploy I:C, RGNNV and SGIV). Comparing with the EcMHC-ⅠA*01 expression levels induced by Zymosan A, Ploy I:C and RGNNV, the effects induced by SGIV and LPS were more significant. Subcellular localization analysis showed that EcMHC-ⅠA*01 localizes throughout the cytoplasm appeared both diffuse and focal intracellular expression pattern. Overexpression of EcMHC-ⅠA*01 inhibited the CPE progression, the mRNA expression of the SGIV related genes (MCP, LITAF, ICP-18 and VP19) and the protein expression of MCP. Meanwhile, qRT-PCR result showed that EcMHC-ⅠA*01 overexpression upregulated the expression of interferon signaling molecules (IFN-γ, ISG56, MDA5 and MXI) and inflammatory cytokines (IL-1ß, IL-6, TNF-α and TRAF6). In addition, our results showed that overexpression of EcMHC-ⅠA*01 promoted the apoptosis of normal fathead minnow (FHM) cells as well as the apoptosis of FHM cells induced by SGIV. However, there was no significant change in the activity of caspase 3 between control group and EcMHC-ⅠA*01 overexpression group, suggesting that EcMHC-ⅠA*01-induced apoptosis may not depend on the caspase 3 pathway. Taken together, these data in our study provide new insights into the role of MHC-I in antiviral immune response and apoptosis in fish.

Grouper TIA-1 functions as a crucial antiviral molecule against nervous necrosis virus infection.

T-cell intracellular antigen (TIA)-1 is a prion-related RNA-binding protein involved in splicing and translational repression, and regulates translation in response to stress conditions by isolating target mRNAs in stress granules (SGs). However, little is known about the potential roles of fish TIA-1 and how it works in viral infection. In this study, the TIA-1 (EcTIA-1) homolog from orange-spotted grouper (Epinephelus coioides) was cloned and characterized. The open reading frame (ORF) sequence of EcTIA-1 encoded a 388 amino acid protein with predicted molecular mass of 42.73 kDa. EcTIA-1 contains three conserved domains of RNA recognition motif (RRM) that may interact with RNA via its second and third RRMs. Overexpression of EcTIA-1 inhibited red-spotted grouper nervous necrosis virus (RGNNV) replication and positively regulated interferon immune response, which was increased by knockdown of EcTIA-1. RGNNV induced formation of SGs in cells with EcTIA-1 overexpression. These results provide a novel insight into understanding the roles of fish TIA-1 in response to RNA viruses.

Anti-lipopolysaccharide factors regulated by Stat, Dorsal, and Relish are involved in anti-WSSV innate immune defense in Macrobrachium nipponense.

Anti-lipopolysaccharide factors (ALF) is an important antimicrobial peptide and critical effector molecule with a broad spectrum of antimicrobial activities in crustaceans. In addition to the previously reported five ALFs (MnALF1-5), another three ALFs [MnALF1, which is different from MnALF1 (ALF02818) that has been reported; MnALF6; and MnALF7] and an isoform of MnALF4 (MnALF4-isoform2) were newly identified from Macrobrachium nipponense in this study. MnALF6 has 134 amino acids and one single nucleotide polymorphism (SNP) in MnALF6 resulted in the change of 107th amino acid from E to D. Intron 1 retention produced longer transcript of MnALF6. The full length of MnALF7 has 691 bp with a 363 bp ORF encoding 120 amino acid protein. Three SNPs in MnALF2 resulted in the conversion of amino acids at positions 70, 73, and 91 from T70I73P91 to K70L73S91. The deletion of 13 bp in MnALF4 resulted in early termination of ORF, resulting in MnALF4-isoform2 with only 98 amino acids. The gDNAs of MnALF1, MnALF2, MnALF5, and MnALF6 contain three exons and two introns, while those of MnALF3 and MnALF7 contain three exons, one known intron, and one unknown intron. The MnALF1-7 in M. nipponense were widely distributed in multiple tissues. After white spot syndrome virus (WSSV) stimulation, the expression levels of MnALF1-7 changed. Knockdown of MnALF1-7 could evidently increase the expression of the envelope protein VP28 and the copy number of WSSV during viral infection. Further studies found that silencing of three transcription factors (Stat, Dorsal, and Relish) in M. nipponense significantly inhibit the synthesis of MnALF1-7 during the process of WSSV challenge. This study adds to the knowledge about the roles of ALFs in the innate immune responses to WSSV infection in M. nipponense.

Comparative evaluation of molecular methods for the quantitative measure of torquetenovirus viremia, the new surrogate marker of immune competence.

BACKGROUND: Torquetenovirus (TTV) viremia is emerging as a promising tool to assess functional immune competence, to predict posttransplant immune-related complications, and eventually to customize immunosuppression. METHODS: In this study, 327 blood samples were tested using two real-time PCR (rtPCR) assays both targeted to the untranslated region of the TTV genome. The first assay was an in-house rtPCR developed by our group, the second one was the recently marketed TTV R-GENE assay. RESULTS: In the validation study, the TTV R-GENE showed good performances in precision and reproducibility, and sensitivity as low as 12 TTV DNA copies/mL, like previously reported for the in-house rtPCR. The Bland-Altman analysis showed that the mean difference between the two methods was -0.3 log copies/mL. In the comparison study, 69% and 72% of samples were detected positive by rtPCR and TTV R-GENE, respectively (94% concordance, κ = 0.88). Performances did not differ between the two rtPCRs by type of TTV group examined. When a newly-developed in-house digital droplet PCR was applied for TTV quantification and used as an alternative method of comparison on 94 samples, the results strongly correlated with those obtained by the two rtPCR methods (99% concordance). CONCLUSION: In summary, all the molecular methods assayed are highly sensitive and accurate in quantitation of TTV DNA in blood samples.

Better detection of Torque teno virus in children with leukemia by metagenomic sequencing than by quantitative PCR.

Torque teno virus (TTV) is a group of chronically persisting viruses with a short circular DNA genome. TTV demonstrates a wide sequence diversity and a large majority of humans are chronically infected by one or more types of TTV. As TTV is ubiquitous, and viral replication correlates with immune status, TTV has been studied as a marker to assess global functional immune competence in transplant recipients. Most studies of the prevalence, amounts, and variation in TTV have been performed using PCR assays. We here present a comparison of the most frequently used quantitative PCR (qPCR) assay for TTV with shotgun metagenomic sequencing for detection and characterization of TTV in a cohort of pediatric cancer patients. The results show that TTV is more common than the qPCR assays indicate, and analysis of the TTV genome sequences indicate that a qPCR with primers and probe designed on a conserved region of the TTV genome may fail to detect some of the TTV strains found in this study.

Phospholipase A2 inhibitor and LY6/PLAUR domain-containing protein PINLYP regulates type I interferon innate immunity.

Type I interferons (IFNs) are the first frontline of the host innate immune response against invading pathogens. Herein, we characterized an unknown protein encoded by phospholipase A2 inhibitor and LY6/PLAUR domain-containing (PINLYP) gene that interacted with TBK1 and induced type I IFN in a TBK1- and IRF3-dependent manner. Loss of PINLYP impaired the activation of IRF3 and production of IFN-ß induced by DNA virus, RNA virus, and various Toll-like receptor ligands in multiple cell types. Because PINLYP deficiency in mice engendered an early embryonic lethality in mice, we generated a conditional mouse in which PINLYP was depleted in dendritic cells. Mice lacking PINLYP in dendritic cells were defective in type I IFN induction and more susceptible to lethal virus infection. Thus, PINLYP is a positive regulator of type I IFN innate immunity and important for effective host defense against viral infection.

MicroRNA-124 Promotes Singapore Grouper Iridovirus Replication and Negatively Regulates Innate Immune Response.

Viral infections seriously affect the health of organisms including humans. Now, more and more researchers believe that microRNAs (miRNAs), one of the members of the non-coding RNA family, play significant roles in cell biological function, disease occurrence, and immunotherapy. However, the roles of miRNAs in virus infection (entry and replication) and cellular immune response remain poorly understood, especially in low vertebrate fish. In this study, based on the established virus-cell infection model, Singapore grouper iridovirus (SGIV)-infected cells were used to explore the roles of miR-124 of Epinephelus coioides, an economically mariculture fish in southern China and Southeast Asia, in viral infection and host immune responses. The expression level of E. coioides miR-124 was significantly upregulated after SGIV infection; miR-124 cannot significantly affect the entry of SGIV, but the upregulated miR-124 could significantly promote the SGIV-induced cytopathic effects (CPEs), the viral titer, and the expressions of viral genes. The target genes of miR-124 were JNK3/p38α mitogen-activated protein kinase (MAPK). Overexpression of miR-124 could dramatically inhibit the activation of NF-κB/activating protein-1 (AP-1), the transcription of proinflammatory factors, caspase-9/3, and the cell apoptosis. And opposite results happen when the expression of miR-124 was inhibited. The results suggest that E. coioides miR-124 could promote viral replication and negatively regulate host immune response by targeting JNK3/p38α MAPK, which furthers our understanding of virus and host immune interactions.

The Roles of Amphibian (Xenopus laevis) Macrophages during Chronic Frog Virus 3 Infections.

Infections by Frog Virus 3 (FV3) and other ranavirus genus members are significantly contributing to global amphibian decline. The Xenopus laevis frog is an ideal research platform upon which to study the roles of distinct frog leukocyte populations during FV3 infections. Frog macrophages (MΦs) are integrally involved during FV3 infection, as they facilitate viral dissemination and persistence but also participate in immune defense against this pathogen. In turn, MΦ differentiation and functionality depend on the colony-stimulating factor-1 receptor (CSF-1R), which is ligated by CSF-1 and iterleukin-34 (IL-34) cytokines. Our past work indicated that X. laevis CSF-1 and IL-34 give rise to morphologically and functionally distinct frog MΦ subsets, and that these CSF-1- and IL-34-MΦs respectively confer susceptibility and antiviral resistance to FV3. Because FV3 targets the frog kidneys and establishes chronic infections therein, presently we examined the roles of the frog CSF-1- and IL-34-MΦs in seeding and maintaining these chronic kidney infections. Our findings indicate that the frog CSF-1-MΦs result in more prominent kidney FV3 infections, which develop into greater reservoirs of lingering FV3 marked by infiltrating leukocytes, fibrosis, and overall immunosuppressive states. Moreover, the antiviral effects of IL-34-MΦs are short-lived and are lost as FV3 infections progress.

Evidence for a Novel Antiviral Mechanism of Teleost Fish: Serum-Derived Exosomes Inhibit Virus Replication through Incorporating Mx1 Protein.

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.

Amphibian (Xenopus laevis) Tadpoles and Adult Frogs Differ in Their Antiviral Responses to Intestinal Frog Virus 3 Infections.

The global amphibian declines are compounded by ranavirus infections such as Frog Virus 3 (FV3), and amphibian tadpoles more frequently succumb to these pathogens than adult animals. Amphibian gastrointestinal tracts represent a major route of ranavirus entry, and viral pathogenesis often leads to hemorrhaging and necrosis within this tissue. Alas, the differences between tadpole and adult amphibian immune responses to intestinal ranavirus infections remain poorly defined. As interferon (IFN) cytokine responses represent a cornerstone of vertebrate antiviral immunity, it is pertinent that the tadpoles and adults of the anuran Xenopus laevis frog mount disparate IFN responses to FV3 infections. Presently, we compared the tadpole and adult X. laevis responses to intestinal FV3 infections. Our results indicate that FV3-challenged tadpoles mount more robust intestinal type I and III IFN responses than adult frogs. These tadpole antiviral responses appear to be mediated by myeloid cells, which are recruited into tadpole intestines in response to FV3 infections. Conversely, myeloid cells bearing similar cytology already reside within the intestines of healthy (uninfected) adult frogs, possibly accounting for some of the anti-FV3 resistance of these animals. Further insight into the differences between tadpole and adult frog responses to ranaviral infections is critical to understanding the facets of susceptibility and resistance to these pathogens.