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SP3 (specificity protein 3) is a transcription factor characterized by three conserved Cys2His2 zinc finger motifs that exert a transregulatory effect by binding to GC boxes, either upregulating or downregulating multiple genes or by co-regulating gene expression in coordination with other proteins. SP3 potentially regulates a series of processes, such as the cell cycle, growth, metabolic pathways, and apoptosis, and plays an important role in antiviral effect. The function of sp3 in fish is poorly understood. In this study, the Sp3a open reading frame was cloned from the orange-spotted grouper, Epinephelus coioides. The full-length open reading frame of Sp3a was 2034 bp, encoding 677 amino acids, with a predicted molecular weight of 72.34 kDa and an isoelectric point of 5.05. Phylogenetically, Sp3a in Epinephelus coioides was the most closely related to Sp3a in the Malabar grouper, Epinephelus malabaricus. RT-qPCR revealed ubiquitous expression of Sp3a in all examined grouper tissues, with no significant differences in expression levels among tissues. A eukaryotic expression vector, pEGFP-Sp3a, was constructed and transfected into grouper spleen (GS) cells. Subcellular localization of Sp3a was observed using an inverted fluorescence microscope. When Spa3 was overexpressed in GS cells, the expression of orange-spotted grouper nerve necrosis virus (RGNNV) genes (CP and RdRp) decreased significantly, indicating that Sp3a significantly inhibited RGNNV replication. siRNA inhibition of Sp3a accelerated the intracellular replication of RGNNV, implying the antiviral effect of Sp3a. Conclusively, our findings contribute to further research on the antiviral capabilities of Sp3a in grouper and other fish. Therefore, our research has potential implications on the development of the aquaculture industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The disease caused by Largemouth bass ranavirus (LMBV) is one of the most severe viral diseases in largemouth bass ( Micropterus salmoides ). It is crucial to evaluate the genetic resistance of largemouth bass to LMBV and develop markers for disease-resistance breeding. In this study, 100 individuals (45 resistant and 55 susceptible) were sequenced and evaluated for resistance to LMBV and a total of 2,579,770 variant sites (SNPs-single-nucleotide polymorphisms (SNPs) and insertions-deletions (InDels)) were identified. A total of 2348 SNPs-InDels and 1018 putative candidate genes associated with LMBV resistance were identified by genome-wide association analyses (GWAS). Furthermore, GO and KEGG analyses revealed that the 10 candidate genes ( MHC II , p38 MAPK , AMPK , SGK1 , FOXO3 , FOXO6 , S1PR1 , IL7R , RBL2 , and GADD45 ) were related to intestinal immune network for IgA production pathway and FoxO signaling pathway. The acquisition of candidate genes related to resistance will help to explore the molecular mechanism of resistance to LMBV in largemouth bass. The potential polymorphic markers identified in this study are important molecular markers for disease resistance breeding in largemouth bass.

Background: Temperature is a crucial environmental determinant for the vitality and development of teleost fish, yet the underlying mechanisms by which they sense temperature fluctuations remain largely unexplored. Transient receptor potential (TRP) proteins, renowned for their involvement in temperature sensing, have not been characterized in teleost fish, especially regarding their temperature-sensing capabilities., Results: In this study, a genome-wide analysis was conducted, identifying a total of 28 TRP genes in the mandarin fish Siniperca chuatsi. These genes were categorized into the families of TRPA, TRPC, TRPP, TRPM, TRPML, and TRPV. Despite notable variations in conserved motifs across different subfamilies, TRP family members shared common structural features, including ankyrin repeats and the TRP domain. Tissue expression analysis showed that each of these TRP genes exhibited a unique expression pattern. Furthermore, examination of the tissue expression patterns of ten selected TRP genes following exposure to both high and low temperature stress indicated the expression of TRP genes were responsive to temperatures changes. Moreover, the expression profiles of TRP genes in response to mandarin fish virus infections showed significant upregulation for most genes after Siniperca chuatsi rhabdovirus, mandarin fish iridovirus and infectious spleen and kidney necrosis virus infection., Conclusions: This study characterized the TRP family genes in mandarin fish genome-wide, and explored their expression patterns in response to temperature stress and virus infections. Our work will enhance the overall understanding of fish TRP channels and their possible functions., (© 2024. The Author(s).)

The inflammasome is a multimeric protein complex that plays a vital role in the defence against pathogens and is therefore considered an essential component of the innate immune system. In this study, the expression patterns of inflammasome genes (NLRC3, ASC, and CAS-1), antiviral genes (IFNγ and MX), and immune genes (IL-1β and IL-18) were analysed in Oreochromis niloticus liver (ONIL) cells following stimulation with the bacterial ligands peptidoglycan (PGN) and lipopolysaccharide (LPS) and infection with TiLV. The cells were stimulated with PGN and LPS at concentrations of 10, 25, and 50 µg/ml. For viral infection, 10 6 TCID 50 of TiLV per ml was used. After LPS stimulation, all seven genes were found to be expressed at specific time points at each of the three doses tested. However, at even higher doses of LPS, NLRC3 levels decreased. Following TiLV infection, all of the genes showed significant upregulation, especially at early time points. However, the gene expression pattern was found to be unique in PGN-treated cells. For instance, NLRC3 and ASC did not show any response to PGN stimulation, and the expression of IFNγ was downregulated at 25 and 50 µg of PGN per ml. CAS-1 and IL-18 expression was downregulated at 25 µg of PGN per ml. At a higher dose (50 µg/ml), IL-1β showed downregulation. Overall, our results indicate that these genes are involved in the immune response to viral and bacterial infection and that the degree of response is ligand- and dose-dependent., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

PACT (interferon-inducible double-stranded RNA-dependent protein kinase activator A) is a cellular protein which can activate PKR in dsRNA-independent manner. However, the role of PACT in fish virus infection remains largely unknown. In this study, a PACT homologue from grouper (Epinephelus coioides)(EcPACT) was cloned and characterized. The open reading frame of EcPACT has a full length of 924 bp and encodes a protein of 307 amino acids with a predicted molecular weight of 33.29 kDa. Similar to mammals, EcPACT contains three dsRBD domains. EcPACT shares 99.67 % homology with E. lanceolatus. Real-time fluorescence quantitative PCR results showed that EcPACT mRNA was widely expressed in all tissues and abundantly expressed in brain, blood, head kidney and kidney. In addition, SGIV and RGNNV infection significantly upregulated the transcript levels of EcPACT. Subcellular localization analysis showed that EcPACT was mainly distributed in the nucleus. Overexpression of EcPACT inhibited the replication of SGIV and RGNNV in vitro and positively regulated the expression of interferon (IFN) and pro-inflammatory factors. The results provide a better understanding of the relationship between PACT and viral infection in fish., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Grouper is one of the most important and valuable mariculture fish in China, with a high economic value. As the production of grouper has increased, massive outbreaks of epidemic diseases have limited the development of the industry. Singapore grouper iridovirus (SGIV) is one of the most serious infectious viral pathogens and has caused huge economic losses to grouper farming worldwide due to its rapid spread and high lethality. To find new strategies for the effective prevention and control of SGIV, we constructed two chimeric DNA vaccines using Lysosome-associated membrane protein 1 (LAMP1) fused with major capsid proteins (MCP) against SGIV. In addition, we evaluated the immune protective effects of vaccines including pcDNA3.1-3HA, pcDNA3.1-MCP, pcDNA3.1-LAMP1, chimeric DNA vaccine pcDNA3.1-MLAMP and pcDNA3.1-LAMCP by intramuscular injection. Our results showed that compared with groups injected with PBS, pcDNA3.1-3HA, pcDNA3.1-LAMP1 or pcDNA3.1-MCP, the antibody titer significantly increased in the chimeric vaccine groups. Moreover, the mRNA levels of immune-related factors in groupers, including IRF3, MHC-I, TNF-α, and CD8, showed the same trend. However, MHC-II and CD4 were significantly increased only in the chimeric vaccine groups. After 28 days of vaccination, groupers were challenged with SGIV, and mortality was documented for each group within 14 days. The data showed that two chimeric DNA vaccines provided 87 % and 91 % immune protection for groupers which were significantly higher than the 52 % protection rate of pcDNA3.1-MCP group, indicating that both forms of LAMP1 chimeric vaccines possessed higher immune protection against SGIV, providing the theoretical foundation for the creation of novel DNA vaccines for fish., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

This study investigated the kinetics of red sea bream iridovirus and host gene expression during infection in rock bream (Oplegnathus fasciatus), a species highly sensitive to the virus. After intraperitoneal injection of the viral solution at 10 4 TCID50/fish, the viral genome copy number in the spleen was 10 4.7 ± 0.2 and 10 5.9 ± 0.4 copies/μg DNA at 3 and 5 days post-injection (dpi), respectively. Using transcriptomic analyses via MiSeq, viral gene transcripts were detected at 3 and 5 dpi. Six genes including RING-finger domain-containing protein and laminin-type epidermal growth factor-like domain genes were significantly expressed at 5 dpi. Further, 334 host genes were differentially expressed compared with those before infection. Genes were clustered into four groups based on their expression profiles. Interferon-stimulated genes were more prevalent in groups showing upregulation at 5 dpi and 3 and 5 dpi. In contrast, the group showing downregulation at 3 dpi included inflammation-related genes, such as granzyme and eosinophil peroxidase genes. Downregulation of certain inflammation-related genes may contribute to the susceptibility of this fish to the virus., (© 2023 John Wiley & Sons Ltd.)

Lymphocystis disease is frequently prevalent and transmissible in various teleost species worldwide due to lymphocystis disease virus (LCDV) infection, causing unsightly growths of benign lymphocystis nodules in fish and resulting in huge economic losses to aquaculture industry. However, the molecular mechanism of lymphocystis formation is unclear. In this study, LCDV was firstly detected in naturally infected flounder ( Paralichthys olivaceus ) by PCR, histopathological, and immunological techniques. To further understand lymphocystis formation, transcriptome sequencing of skin nodule tissue was performed by using healthy flounder skin as a control. In total, RNA-seq produced 99.36%-99.71% clean reads of raw reads, of which 91.11%-92.89% reads were successfully matched to the flounder genome. The transcriptome data showed good reproducibility between samples, with 3781 up-regulated and 2280 down-regulated differentially expressed genes. GSEA analysis revealed activation of Wnt signaling pathway, Hedgehog signaling pathway, Cell cycle, and Basal cell carcinoma associated with nodule formation. These pathways were analyzed to interact with multiple viral infection and tumor formation pathways. Heat map and protein interaction analysis revealed that these pathways regulated the expression of cell cycle-related genes such as ccnd1 and ccnd2 through key genes including ctnnb1 , lef1 , tcf3 , gli2 , and gli3 to promote cell proliferation. Additionally, cGMP-PKG signaling pathway, Calcium signaling pathway, ECM-receptor interaction, and Cytokine-cytokine receptor interaction associated with nodule formation were significantly down-regulated. Among these pathways, tnfsf12 , tnfrsf1a , and tnfrsf19 , associated with pro-apoptosis, and vdac2 , which promotes viral replication by inhibiting apoptosis, were significantly up-regulated. Visual analysis revealed significant down-regulation of cytc , which expresses the pro-apoptotic protein cytochrome C, as well as phb and phb2 , which have anti-tumor activity, however, casp3 was significantly up-regulated. Moreover, bcl9 , bcl11a , and bcl-xl , which promote cell proliferation and inhibit apoptosis, were significantly upregulated, as were fgfr1 , fgfr2 , and fgfr3 , which are related to tumor formation. Furthermore, RNA-seq data were validated by qRT-PCR, and LCDV copy numbers and expression patterns of focused genes in various tissues were also investigated. These results clarified the pathways and differentially expressed genes associated with lymphocystis nodule development caused by LCDV infection in flounder for the first time, providing a new breakthrough in molecular mechanisms of lymphocystis formation in fish., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Zhang, Sheng, Tang, Xing, Chi and Zhan.)

Protein kinase C (PKC) constitutes the main signal transduction pathway, and participates in the signal pathway of cell proliferation and movement in mammals. In this study, PKC-ɑ was obtained from Epinephelus coioides, an important marine fish cultivated in the coastal areas of southern China and Southeast Asia. The full length cDNA of PKC-ɑ was 3362 bp in length containing a 23 bp 5'UTR, a 1719 bp 3'UTR, and a 1620 bp open reading frame encoding 539 amino acids. It contains three conservative domains including protein kinase C conserved region 2 (C2), Serine/Threonine protein kinases, catalytic domain (S_TKc) and ser/thr-type protein kinases (S_TK_X). Its mRNA can be detected in all 11 tissues examined of E. coioides, and the expression was significantly upregulated response to Singapore grouper iridovirus (SGIV) infection, one of the important pathogens of marine fish. Upregulated E. coioides PKC-ɑ significantly inhibited the activation of nuclear factor kappa-B (NF-κB) and activator protein-1 (AP-1), and SGIV-induced cell apoptosis. The results indicated that the PKC-ɑ may play an important role in pathogenic stimulation., (Copyright © 2023. Published by Elsevier Ltd.)

Background: Megalocytiviruses (MCV) are double-stranded DNA viruses that infect fish. Two species within the genus are epidemiologically important for fish farming: red sea bream iridovirus (RSIV) and infectious spleen and kidney necrosis virus (ISKNV). The objective of this work was to study regions that allow the differentiation and correct diagnosis of RSIV and ISKNV., Methods: The regions ORF450L, ORF342L, ORF077, and the intergenic region between ORF37 and ORF42R were sequenced and compared with samples from the database., Results: The tree constructed using the sequencing of the PCR product Megalocytivirus. ORF077 separated the three major clades of MCV. RISV genotypes were well divided, but not ISKNV. All qPCRs tests showed acceptable repeatability values, that is, less than 5%., Conclusion: Two qPCRs for ISKNV detection and two for RSIV were considered suitable for use in the diagnosis and typing of MCV. The results of this study demonstrate the importance of an accurate evaluation of methodologies for the differentiation of MCV., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Iridoviruses are large DNA viruses which cause great economic losses to the aquaculture industry and serious threats to ecological diversity worldwide. Singapore grouper iridovirus (SGIV), a novel member of the genus Ranavirus , causes high mortality in grouper aquaculture. Previous work on genome annotation demonstrated that SGIV contained numerous uncharacterized or hypothetical open reading frames (ORFs), whose functions remained largely unknown. Here, we reported that the protein encoded by SGIV ORF131R (VP131) was localized predominantly within the endoplasmic reticulum (ER). Ectopic expression of GFP-VP131 significantly enhanced SGIV replication, while VP131 knockdown decreased viral infection in vitro , suggesting that VP131 functioned as a proviral factor during SGIV infection. Overexpression of GFP-VP131 inhibited the interferon (IFN)-1 promoter activity and mRNA level of IFN-related genes induced by poly(I:C), Epinephelus coioides cyclic GMP/AMP synthase (EccGAS)/stimulator of IFN genes (EcSTING), TANK-binding kinase 1 (EcTBK1), or melanoma differentiation-associated gene 5 (EcMDA5), whereas such activation induced by mitochondrial antiviral signaling protein (EcMAVS) was not affected. Moreover, VP131 interacted with EcSTING and degraded EcSTING through both the autophagy-lysosome pathway and ubiquitin-proteasome pathway, and targeted for the K63-linked ubiquitination. Of note, we also found that EcSTING significantly accelerated the formation of GFP-VP131 aggregates in co-transfected cells. Finally, GFP-VP131 inhibited EcSTING- or EcTBK1-induced antiviral activity upon red-spotted grouper nervous necrosis virus (RGNNV) infection. Together, our results demonstrated that the SGIV VP131 negatively regulated the IFN response by inhibiting EcSTING-EcTBK1 signaling for viral evasion. IMPORTANCE STING has been identified as a critical factor participating in the innate immune response which recruits and phosphorylates TBK1 and IFN regulatory factor 3 (IRF3) to induce IFN production and defend against viral infection. However, viruses also distort the STING-TBK1 pathway to negatively regulate the IFN response and facilitate viral replication. Here, we reported that SGIV VP131 interacted with EcSTING within the ER and degraded EcSTING, leading to the suppression of IFN production and the promotion of SGIV infection. These results for the first time demonstrated that fish iridovirus evaded the host antiviral response via abrogating the STING-TBK1 signaling pathway.

Persistent virus infections are achieved when the intricate balance of virus replication, host-cell division and successful immune evasion is met. The genomes of persistent DNA viruses are either maintained as extrachromosomal episomes or can integrate into the host genome. Common to both these strategies of persistence is the chromatinisation of viral DNA by cellular histones which, like host DNA, are subject to epigenetic modification. Epigenetic repression of viral genes required for lytic replication occurs, while genes required for latent or persistent infection are maintained in an active chromatin state. Viruses utilise host-cell chromatin insulators, which function to maintain epigenetic boundaries and enforce this strict transcriptional programme. Here, we review insulator protein function in virus transcription control, focussing on CCCTC-binding factor (CTCF) and cofactors. We describe CTCF-dependent activities in virus transcription regulation through epigenetic and promoter-enhancer insulation, three-dimensional chromatin looping and manipulation of transcript splicing., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Approximately 170 RNA modifications have been identified and these are critical for determining the fate and function of cellular RNAs. Similar to human transcripts, viral RNAs possess an extensive RNA modification landscape. While initial efforts largely focused on investigating the RNA modification landscape in the context of RNA virus infection, a growing body of work has explored the impact of RNA modifications on DNA virus biology. These studies have revealed roles for RNA modifications in DNA virus infection, including gene regulation and viral pathogenesis. In this review, we will discuss the current knowledge on how RNA modifications impact DNA virus biology., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The DNA sensor cGAS detects cytosolic DNA and instigates type I interferon (IFN) expression. Recent studies find that cGAS also localizes in the nucleus and binds the chromatin. Despite the mechanism controlling nuclear cGAS activation is well elucidated, whether nuclear cGAS participates in DNA sensing is unclear. Here, we report that herpes simplex virus 1 (HSV-1) infection caused the release of cGAS from the chromatin into the nuclear soluble fraction. Like its cytosolic counterpart, the leaked nuclear soluble cGAS also sensed viral DNA, produced cGAMP, and induced mRNA expression of type I IFN and interferon-stimulated genes. Consistently, the nuclear soluble cGAS limited HSV-1 infection. Furthermore, enzyme-deficient mutation (D307A) or cGAS inhibitor RU.251 abolished nuclear cGAS-mediated innate immune responses, suggesting that enzymatic activity is also required for nuclear soluble cGAS. Taken all together, our study demonstrates that nuclear soluble cGAS acts as a nuclear DNA sensor detecting nuclear-replicating DNA viruses., (© 2022. The Author(s).)

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-β 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., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The genus Megalocytivirus includes viruses known to cause significant disease in aquacultured fish stocks. Herein, we report the complete genome sequences of two megalocytiviruses (MCVs) isolated from diseased albino rainbow sharks Epalzeorhynchos frenatum reared on farms in the United States in 2018 and 2019. Histopathological examination revealed typical megalocytivirus microscopic lesions (i.e., basophilic cytoplasmic inclusions) that were most commonly observed in the spleen and kidney. Transmission electron microscopic examination of spleen and kidney tissues from specimens of the 2018 case revealed hexagonally shaped virus particles with a mean diameter of 153 ± 6 nm (n = 20) from opposite vertices and 131 ± 5 nm (n = 20) from opposite faces. Two MCV-specific conventional PCR assays confirmed the presence of MCV DNA in the collected samples. Full genome sequencing of both 2018 and 2019 Epalzeorhynchos frenatus iridoviruses (EFIV) was accomplished using a next-generation sequencing approach. Phylogenomic analyses revealed that both EFIV isolates belong to the infectious spleen and kidney necrosis virus (ISKNV) genotype within the genus Megalocytivirus. This study is the first report of ISKNV in albino rainbow sharks., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cell survival is based on the stability of intracellular state. It was well known that biochemical reactions in cells require specific intracellular environments, such as pH and calcium concentration. While the mechanism of stabilizing the intracellular environment is complex and far from clear. In this study, a Sma and Mad related protein 5 gene (LvSmad5) of Litopenaeus vannamei was cloned. LvSmad5 was located to both cytoplasm and nucleus. And subcellular localization of LvSmad5 was responsed to the changing of cells internal and external environment. Besides, it was found that subcellular localization of LvSmad5 was also regulated by unfolded protein response. Moreover, it was proved that nucleic localization of LvSmad5 could significantly increase the white spot syndrome virus (WSSV) infection in shrimp, and knockdown expression of LvSmad5 decreased the cumulative mortality of WSSV infection shrimp. Further investigation revealed that cytoplasm LvSmad5 could interplay with shrimp hexokinase 1, and contribute to glycolysis. These results indicated that LvSmad5 played a role in L. vannamei environmental stress response, and was used by WSSV for its replication., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Multiple novel circular replication-associated protein (Rep)-encoding single stranded (CRESS) DNA viruses have been extensively identified in the feces of humans and animals. Here, we first detected CRESS DNA virus (named Horse-CRESS DNA-like virus, HCLV) in two fecal samples from 10 imported thoroughbred (TB) horses in the customs quarantine station in North Xinjiang province, China. Additionally, we found that this virus was not detected in local breeds (LBs) (0/41) and was found only in imported TB horses (2/73). We obtained the whole-genome sequences of four viruses (HCLV ALSK-3-4, ALSK-13-10, CJ-1-2, and CJ-13-1). Unlike Circovirus and Cyclovirus, whose genome sequences have 1700 to 2100 nucleotides (nt), these HCLVs have circular genome with 3503, 3504, 3485, 3491 nt, respectively and five major ORFs. The ORF1 gene encodes the Rep protein in HCLVs. Furthermore, the Rep protein of the four HCLVs share 23.3-84.8%, 21.6-27.4%, 23.7-27.2% amino acid identity with the corresponding reference viruses of Kirkoviruses, genus Circovirus, and genus Cyclovirus, respectively. Moreover, RCR domain, P-loop NTPase domains, and nonanucleotide motif (TAGTATTAC) of the HCLVs are similar to Circovirus and Cyclovirus. Phylogenetic analysis showed that the virus was grouped together with members in Kirkoviruses. These results suggest the HCLV probably entered Xinjiang province via the international trade of horses.

Background: Frog Virus 3 (FV3) is a large dsDNA virus belonging to Ranaviruses of family Iridoviridae . Ranaviruses infect cold-blood vertebrates including amphibians, fish and reptiles, and contribute to catastrophic amphibian declines. FV3 has a genome at ~105 kb that contains nearly 100 coding genes and 50 intergenic regions as annotated in its reference genome. Previous studies have mainly focused on coding genes and rarely addressed potential non-coding regulatory role of intergenic regions., Results: Using a whole transcriptomic analysis of total RNA samples containing both the viral and cellular transcripts from FV3-infected frog tissues, we detected virus-specific reads mapping in non-coding intergenic regions, in addition to reads from coding genes. Further analyses identified multiple cis -regulatory elements ( CREs ) in intergenic regions neighboring highly transcribed coding genes. These CREs include not only a virus TATA-Box present in FV3 core promoters as in eukaryotic genes, but also viral mimics of CREs interacting with several transcription factors including CEBPs, CREBs, IRFs, NF-κB, and STATs, which are critical for regulation of cellular immunity and cytokine responses. Our study suggests that intergenic regions immediately upstream of highly expressed FV3 genes have evolved to bind IRFs, NF-κB, and STATs more efficiently. Moreover, we found an enrichment of putative microRNA (miRNA) sequences in more than five intergenic regions of the FV3 genome. Our sequence analysis indicates that a fraction of these viral miRNAs is targeting the 3'-UTR regions of Xenopus genes involved in interferon (IFN)-dependent responses, including particularly those encoding IFN receptor subunits and IFN-regulatory factors (IRFs)., Conclusions: Using the FV3 model, this study provides a first genome-wide analysis of non-coding regulatory mechanisms adopted by ranaviruses to epigenetically regulate both viral and host gene expressions, which have co-evolved to interact especially with the host IFN response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tian, Khwatenge, Li, De Jesus Andino, Robert and Sang.)

Objectives: An association between costimulatory molecule gene polymorphisms and viral infection after hematopoietic stem cell transplantation may be related to clinical outcomes, especially acute graft-versus-host disease. Cytotoxic T-lymphocyte antigen 4 has been suggested as a crucial negative regulator of the immune system. In this study, our objective was to investigate the association between cytotoxic T-lymphocyte antigen-4 gene polymorphisms (including -1722 T/C, -1661 A/G, -318 C/T, and +49 A/G) and torque teno virus infection after hematopoietic stem cell transplantation in patients with and without acute graft-versus-host disease., Materials and Methods: Our study included 71 recipients. We evaluated cytotoxic T-lymphocyte antigen 4 gene polymorphisms using the polymerase chain reaction-restriction fragment length polymorphism method., Results: Our results showed that the GG genotype of the cytotoxic T-lymphocyte antigen 4 +49 A/G was significantly more frequent in transplanted patients infected with torque teno virus, whereas the AG genotype was more common in transplanted patients who did not have this infection. In addition, the -1661 AA and GA genotypes and -318 TC genotypes were significantly more frequent in transplanted patients infected with the virus and who had low-grade (grades I and II) acute graft-versus-host disease. Among those with grade I graft-versus-host disease, the GG genotype of the cytotoxic T-lymphocyte antigen 4+49 A/G was more frequent in transplanted patients with torque teno virus infection, whereas the AG genotype was higher in transplanted patients who did not have this infection., Conclusions: This is the first report indicating that cytotoxic T-lymphocyte antigen 4 gene polymorphism may be implicated in prevalence of torque teno virus infection after stem cell transplant. Further larger studies and evaluation of other costimulatory molecules are suggested.

IFI16, hnRNPA2B1, and nuclear cGAS are nuclear-located DNA sensors that play important roles in initiating host antiviral immunity and modulating tumorigenesis. IFI16 triggers innate antiviral immunity, inflammasome, and suppresses tumorigenesis by recognizing double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), damaged nuclear DNA, or cooperatively interacting with multiple tumor suppressors such as p53 and BRCA1. hnRNPA2B1 initiates interferon (IFN)-α/β production and enhances STING-dependent cytosolic antiviral signaling by directly binding viral dsDNA from invaded viruses and facilitating N 6 -methyladenosine (m 6 A) modification of cGAS, IFI16, and STING mRNAs. Nuclear cGAS is recruited to double-stranded breaks (DSBs), suppresses DNA repair, and promotes tumorigenesis. This review briefly describes the nuclear functions of IFI16, hnRNPA2B1, and cGAS, and summarizes the transcriptional, post-transcriptional, and post-translational regulation of these nuclear DNA sensors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Yuan and Ma.)

Torque teno virus (TTV) is a commensal human virus observed as a circular single-negative-strand DNA molecule in various tissues and biological samples, notably in blood serum and lymphocytes. TTV has no apparent clinical significance, although it might be very useful as a prospective tool for gene delivery or as an epidemiological marker. Human populations are ubiquitously infected with TTV; the prevalence may reach 100%. The majority of babies become spontaneously infected with TTV, so that by the end of the first year of life, the prevalence reaches 'adult' values. TTV positivity in healthy early infancy and the presence of TTV in umbilical cord blood samples have been reported. The mechanism of infection and the dynamics of TTV prevalence in infants with age remain understudied. Meanwhile, the potential diagnostic and prognostic value of TTV as a marker deserves special attention and study, along with the possibility, causes and consequences of placental transmission of TTV under normal or pathological conditions.

Since the mechanisms by which cellular factors modulate replication of the shrimp viral pathogen white spot syndrome virus (WSSV) are still largely unknown, here we consider the sirtuins, a family of NAD + -dependent protein deacetylases that are known to function as regulatory factors that activate or suppress viral transcription and replication in mammals. In particular, we focus on SIRT1 by isolating and characterizing LvSIRT1 from white shrimp (Litopenaeus vannamei) and investigating its involvement in WSSV infection. DsRNA-mediated gene silencing led to the expression of WSSV genes and the replication of genomic DNAs being significantly decreased in LvSIRT1-silenced shrimp. The deacetylase activity of LvSIRT1 was significantly induced at the early stage (2 hpi) and the genome replication stage (12 hpi) of WSSV replication, but decreased at the late stage of WSSV replication (24 hpi). Treatment with the SIRT1 activator resveratrol further suggested that LvSIRT1 activation increased the expression of several WSSV genes (IE1, VP28 and ICP11). Lastly, we used transfection and dual luciferase assays in Sf9 insect cells to show that while the overexpression of LvSIRT1 facilitates the promoter activity of WSSV IE1, this enhancement of WSSV IE1 expression is achieved by a transactivation pathway that is NF-κB-independent., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The DNA sensor cGMP-AMP synthase (cGAS) senses cytosolic microbial or self DNA to initiate a MITA/STING-dependent innate immune response. cGAS is regulated by various posttranslational modifications at its C-terminal catalytic domain. Whether and how its N-terminal unstructured domain is regulated by posttranslational modifications remain unknown. We identified the acetyltransferase KAT5 as a positive regulator of cGAS-mediated innate immune signaling. Overexpression of KAT5 potentiated viral-DNA-triggered transcription of downstream antiviral genes, whereas a KAT5 deficiency had the opposite effects. Mice with inactivated Kat5 exhibited lower levels of serum cytokines in response to DNA virus infection, higher viral titers in the brains, and more susceptibility to DNA-virus-induced death. Mechanistically, KAT5 catalyzed acetylation of cGAS at multiple lysine residues in its N-terminal domain, which promoted its DNA-binding ability. Our findings suggest that KAT5-mediated cGAS acetylation at its N terminus is important for efficient innate immune response to DNA virus., Competing Interests: The authors declare no competing interest.

Decapod iridescent virus 1 (DIV1) results in severe economic losses in shrimp aquaculture. However, little is known about the physiological effect of DIV1 infection on the host. In this study, we found that the lethal dose 50 of DIV1-infected Litopenaeus vannamei after 48, 72, 96, and 156 h were 4.86 × 10 6 , 5.07 × 10 5 , 2.13 × 10 5 , and 2.38 × 10 4 copies/μg DNA, respectively. In order to investigate the mechanisms of DIV1 infection, a comparative transcriptome analysis of hemocytes from L. vannamei , infected or not with DIV1, was conducted. The BUSCO analysis showed that the transcriptome was with high completeness (complete single-copy BUSCOs: 57.3%, complete duplicated BUSCOs: 41.1%, fragmentation: 0.8%, missing: 0.8%). A total of 168,854 unigenes were assembled, with an average length of 601 bp. Based on homology searches, Kyoto Encyclopedia of Genes and Genomes (KEGG), gene ontology (GO), and cluster of orthologous groups of proteins (KOG) analysis, 62,270 (36.88%) unigenes were annotated. Among them, 1,112 differentially expressed genes (DEGs) were identified, of which 889 genes were up-regulated and 223 genes were down-regulated after DIV1 infection. These genes were mainly annotated to the major metabolic processes such as fructose and mannose metabolism, carbon metabolism, and inositol phosphate metabolism. Among these metabolic pathways, the triosephosphate isomerase ( TPI ) family was the most eye-catching DEG as it participates in several metabolic processes. Three types of TPI, LvTPI-like, LvTPI-Blike , and LvTPI-Blike1 , were obtained for gene silencing by RNA interference. The results showed that LvTPI-like and LvTPI-Blike1 silencing caused a high mortality rate among L. vannamei . However, LvTPI-like and LvTPI-Blike silencing reduced DIV1 replication in DIV1-infected L. vannamei . All the results indicated that TPI-like genes play an important role during DIV1 infection, which provides valuable insight into the infection mechanism of DIV1 in shrimp and may aid in preventing viral diseases in shrimp culture., (Copyright © 2020 Liao, Wang, Wang, Qin, Hu, Wang, Sun and Zhang.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In mammals, casitas B-lineage lymphoma (CBL) family proteins, a RING-type E3 ubiquitin ligase, are involved in many signal transduction pathways. However, the functions of CBL in invertebrates are not well elucidated. In this study, Sp-CBL containing CBL-N, CBL-2, CBL-3 and RING domains was identified in mud crab Scylla paramamosain. Sp-CBL was widely expressed in all tissues tested and found to be significantly up-regulated in the hemocytes of mud crab challenged by white spot syndrome virus (WSSV). The RNA interference of Sp-CBL increased the copy number of WSSV and declined the apoptosis rate of hemocytes. In addition, Sp-CBL could affect the activities of caspase 3 and the mitochondrial membrane potential. Taken together, the results of this study revealed that Sp-CBL could restrict WSSV proliferation through enhancing the apoptosis of the hemocytes, which would provide a novel insight into the anti-viral response in the innate immunity system of mud crab., (Copyright © 2019. Published by Elsevier Ltd.)

The Kruppel-like factor 6 (KLF6) is a member of Kruppel-like factor family, which belong to the Zinc finger family of transcription factors that mediates various cellular processes, such as proliferation, differentiation, development, and programmed cell death. Peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily and they regulate numerous genes through ligand-dependent transcriptional activation and repression. In this study, we focus on the role of KLF6 gene in virus infection and the regulation of KLF6 on PPAR-δ in orange-spotted grouper (Epinephelus coioides). The ORF sequence of EcKLF6 was 846 bp, encoding a polypeptide of 282 amino acids with three conserved Zinc finger (type Cys 2 -His 2 ) domain in the C-terminal region. Basing on the detection of the mRNA levels of viral genes, western blotting of MCP protein, and morphological CPEs, we found that the overexpression of EcKLF6 suppressed the replication of Singapore grouper iridovirus (SGIV), exerting its antiviral activity against fish virus. Moreover, promoter analysis was performed to investigate whether EcKLF6 was a regulator of EcPPAR-δ. The luciferase reporter assay and real time PCR results indicated a negative regulatory role of EcKLF6 on EcPPAR-δ transcription in grouper. Further experimental analysis shows that the potential EcKLF6 binding sites may locate in the EcPPAR-δ-4-M3 (+133 to +154) and EcPPAR-δ-4-M4 (+354 to +368) region of the EcPPAR-δ promoter. Electrophoretic mobile shift assays (EMSAs) verified that EcKLF6 interacted with the binding site of the EcPPAR-δ-4-M4 promoter region. In addition, we also found that KLF6 promotes inflammatory responses in GS cells. Considering that KLF6 and PPAR-δ play opposite roles in regulating inflammatory responses, we speculated the promoting effect of KLF6 on inflammatory response may be related to its negative regulation on EcPPAR-δ. In conclusion, the present study provides the first evidence of the negative regulation of EcPPAR-δ transcription by EcKLF6 and contributes to a better understanding of the transcriptional mechanisms of EcKLF6 in fish., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Over the last decade, innate immune priming has been evidenced in many invertebrate phyla. If mechanistic models have been proposed, molecular studies aiming to substantiate these models have remained scarce. We reveal here the transcriptional signature associated with immune priming in the oyster Crassostrea gigas Oysters were fully protected against Ostreid herpesvirus 1 (OsHV-1), a major oyster pathogen, after priming with poly(I·C), which mimics viral double-stranded RNA. Global analysis through RNA sequencing of oyster and viral genes after immune priming and viral infection revealed that poly(I·C) induces a strong antiviral response that impairs OsHV-1 replication. Protection is based on a sustained upregulation of immune genes, notably genes involved in the interferon pathway and apoptosis, which control subsequent viral infection. This persistent antiviral alert state remains active over 4 months and supports antiviral protection in the long term. This acquired resistance mechanism reinforces the molecular foundations of the sustained response model of immune priming. It further opens the way to applications (pseudovaccination) to cope with a recurrent disease that causes dramatic economic losses in the shellfish farming industry worldwide. IMPORTANCE In the last decade, important discoveries have shown that resistance to reinfection can be achieved without a functional adaptive immune system, introducing the concept of innate immune memory in invertebrates. However, this field has been constrained by the limited number of molecular mechanisms evidenced to support these phenomena. Taking advantage of an invertebrate species, the Pacific oyster ( Crassostrea gigas ), in which we evidenced one of the longest and most effective periods of protection against viral infection observed in an invertebrate, we provide the first comprehensive transcriptomic analysis of antiviral innate immune priming. We show that priming with poly(I·C) induced a massive upregulation of immune-related genes, which control subsequent viral infection, and it was maintained for over 4 months after priming. This acquired resistant mechanism reinforces the molecular foundations of the sustained response model of immune priming. It opens the way to pseudovaccination to prevent the recurrent diseases that currently afflict economically or ecologically important invertebrates., (Copyright © 2020 Lafont et al.)

The purpose of this study was to determine whether timing of the initiating event of spontaneous labor (either uterine contractions with intact fetal membranes or rupture of membranes prior to labor (PROM)) is associated with maternal viral infection. It was a prospective case control study of women with either spontaneous labor or PROM occurring < 37 weeks' gestation ("cases") or at term ("controls"). An initial unbiased screen for viruses was performed with next-generation sequencing (NGS) in serum pooled from eight cases delivered by C/S and represents a range of gestational ages, membrane rupture status, and presence or absence of chorioamnionitis. Custom PCR was used to query individual patient samples from the original cohort. The NGS screen generated 15 million reads. Seven unique viral sequences were detected in two cases, all identified as torque teno virus (TTV), an ubiquitous DNA anellovirus of no known pathogenicity. Using nested and semi-nested PCR, sera from 72 patients (47 cases and 25 matched controls, stratified by ROM status) were screened for the 3 subtypes of anelloviruses (TTV, TTMDV, or TTMV). These were found in 43/47 cases (91%) and 16/25 controls (64%) (p = 0.012, OR = 5.9 (95% CI = 1.4-29.9)). In logistic regression, pregnant women with at least one type of anellovirus were more likely to experience preterm labor than those with no anellovirus (p = 0.03, aOR = 4.6, CI = 1.2-18.7). Among women experiencing a spontaneous initiating event of labor, TTV virus was more likely to be present in the serum of preterm than term patients. TTV may have a role in determining the timing of parturition.

Hepcidin is a liver-derived peptide hormone that is related to iron balance and immunity in humans. However, its function in Siniperca chuatsi has not been well elucidated. In this study, we analyzed the expression and function of the S. chuatsi hepcidin ( Sc-hep ) gene. Sc-hep was specifically expressed in the liver and appeared to be one of the most highly expressed genes in the liver. After spleen and kidney necrosis virus (ISKNV) infection and lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (Poly I:C) stimulation, the expression of Sc-hep in the liver increased by approximately 110, 6500, and 225 times, respectively. After ferrous sulfate (FS) injection, the expression of Sc-hep in the liver increased approximately 520-fold. We found that miR-19c-5p could inhibit Sc-hep expression. Five CpG dinucleotides distributed in the promoter region showed no differential methylation between the liver and the stomach, both presenting high methylation rates. After FS or LPS injection, the expression of three iron balance-related genes ( FPN1 , TFR1 , and FTN ) and five immune-related cytokine genes ( IL-1 β, IL8 , TNF- α, TLR22 , and SOCS3 ) significantly changed. These results indicate that Sc-hep participates in the regulation of iron balance and plays an important role in the immune system. Sc-hep increased approximately 52-fold when mandarin fish were domesticated with artificial diets. Sc-hep might be used as a real-time biomarker of mandarin fish liver because its expression markedly varies under different physiological conditions.

White spot syndrome virus (WSSV) is the causative agent of a shrimp disease that has caused huge global economic losses. Although its pathogenesis remains poorly understood, it has been reported that in the shrimp immune cells (hemocytes) targeted by WSSV, the virus triggers both the Warburg effect and glutamine metabolism at the WSSV genome replication stage (12 h post infection). Glutamine metabolism follows two pathways: an oxidative pathway mediated by α-KGDH (α-ketoglutarate dehydrogenase) and an alternative reductive pathway mediated by IDH1 and IDH2 (isocitrate dehydrogenase 1 and 2). Here we used isotopically labeled glutamine ([U- 13 C]glutamine and [1- 13 C]glutamine) as metabolic tracers to show that, at the replication stage, both the oxidative and reductive glutamine metabolic pathways were activated. We further show that the mRNA expression levels of α-KGDH and IDH1 were increased in WSSV-infected shrimps and that silencing of α-KGDH, IDH1, and IDH2 with their respective dsRNAs led to a decrease in WSSV gene expression and WSSV replication. Taken together, our findings provide new evidence for WSSV-induced metabolic reprogramming in hemocytes and demonstrate its importance in virus replication.

The TRIpartite Motif (TRIM) proteins are known to play key roles in cell differentiation, apoptosis, development, autophagy and innate immunity. In the present study, a TRIM9 homolog (named LvTRIM9) was identified from the transcriptome of the Pacific whiteleg shrimp Litopenaeus vannamei. The deduced amino acid sequence of LvTRIM9 possessed typical features of TRIMs, consisting of a RING domain, two B-boxes, a coiled-coil domain, a FN3 domain, and a SPRY domain. The transcript of LvTRIM9 was detected in most tissues of the shrimp. Its expression level was obviously up-regulated at 3, 12 and 24 h post white spot syndrome virus (WSSV) infection. Knockdown of LvTRIM9 gene expression by double-strand RNA mediated interference could lead to a decrease of virus copy number in WSSV-infected shrimp. Yeast two-hybrid analysis showed that LvTRIM9 could directly interact with beta-transducin repeat-containing protein of shrimp (Lvβ-TrCP), an inhibitor of NF-κB pathway. Meanwhile, knockdown of LvTRIM9 could also up-regulate the expression levels of LvRelish and downstream production of antimicrobial peptides in the intestine of shrimp. These data indicated that WSSV might hijack the LvTRIM9 for its propagation through inhibition of NF-κB pathway and downstream antimicrobial peptides production via interaction of LvTRIM9 with Lvβ-TrCP in shrimp. The study improved our understanding about the impact of E3 ubiquitin ligases on the innate immune signaling pathway of shrimp and its role during WSSV infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) as short noncoding RNAs play important regulatory roles in diverse biological processes by degrading the target mRNAs, and could be delivered by exosomes. WSSV is a highly pathogenic and prevalent virus, and has brought high mortality of P. clarkia. Till present, no studies focus on the miRNAs changes in exosomes during WSSV infection. To understand the different virulence of WSSV on miRNAs expression in P. clarkia hemolymph exosome, the deep sequencing was performed to compare the small RNA libraries from the hemolymph exosome of P. clarkia individuals with or without WSSV infections. From the TEM observations, NTA and Western Blot analysis, the extracted exosomes were well identified with classic characteristics. The 209 conserved miRNAs and 250 novel miRNAs were identified from the small RNA libraries. In response to WSSV infection, there were about 98 miRNAs significantly up-regulated and 59 miRNAs significantly down-regulated. The target genes prediction, GO and KEGG enrichment analysis revealed that some target genes of P. clarkia miRNAs were grouped mainly into the categories of biological regulation, immune system process, signal pathway and other more functions. This is the first report of comprehensive identification of P. clarkia hemolymph exosome miRNAs being differentially regulated in response to WSSV infection. These results will help to understand the hemolymph exosome miRNAs response to different virulence WSSV infection., (Copyright © 2018. Published by Elsevier Ltd.)

Little is known about the role of genetic variation in the genes for cytokines and susceptibility to viral infection especially torque teno virus (TTV) following allogeneic hematopoietic stem cell transplantation. In this study, the association between interleukin-12, interleukin-17, interleukin-10 (IL-12,-17,-10) and tumor necrosis factor-α (TNF-α) polymorphisms was evaluated in patients with TTV infection who underwent allogeneic hematopoietic stem cell transplantation from South of Iran. The single nucleotide polymorphisms in the cytokine genes including IL-12 (-1188A/C), IL-17 (-197G/A), IL-10 (-1082G/A, -819C/T and -592C/A) and TNF-α (-308 G/A) were analyzed by PCR-RFLP methods. While our results did not show any association between IL-17, IL-12 and IL-10 (-819C/T and -1082G/A) polymorphisms and TTV infection status, heterozygote genotype of IL-10 (-592C/A) had direct correlation with TTV infection and A allele of TNF-α (-308G/A) showed a protective effect against TTV infection (P = 0.05 and P = 0.025, respectively). Within the group of patients who experienced acute graft-versus-host disease, the AA genotype and the A allele of IL-17 (-197 G/A) were significantly higher in non-infected patients compared to infected ones (P = 0.024 and P = 0.057, respectively). It was also observed that among infected patients, the GG genotype of IL-17 and AA genotype of TNF-α were significantly increased in hematopoietic stem cell transplanted patients with low grade (grade I+II) acute graft-versus-host disease compared to high grade (grade III and IV) disease (P = 0.056 and P = 0.056, respectively). Taken together, genetic variation of IL-10 (-592C/A) and TNF-α (-308G/A) genes might be associated with susceptibility to TTV infection post hematopoietic stem cell transplantation. Keywords: TNF-α; interleukins; torque teno virus (TTV); hematopoietic stem cell transplantation (HSCT); graft versus host disease (GvHD).

MicroRNAs (miRNAs) are important posttranscriptional regulators. They play an important role in the antiviral innate immunity of invertebrates. In the present study, high-throughput small RNAs Illumina sequencing systems were carried out to identify differentially expressed miRNAs (DEMs) in the gills of Procambarus clarkii, which was challenged with white spot syndrome virus (WSSV). Our results identified 11,617 known and 6 novel miRNAs in normal group (NG) and WSSV-challenged group (WG) small RNA libraries. Additionally, 27 DEMs were shown to participate in the antiviral innate immunity of P. clarkii and were significantly upregulated or downregulated. In addition, the results of the KEGG pathway prediction of the DEMs target genes showed that putative target genes of these 27 DEMs were related mainly to the RNA transport pathway, tight junction pathway, mRNA surveillance pathway, regulation actin cytoskeleton pathway, focal adhesion pathway, and MAPK signaling pathway. These results provide important information for future studies about the antiviral innate immunity of crustaceans., (Copyright © 2018. Published by Elsevier Ltd.)

Bcl-2 family proteins play essential roles in modulating immune response and controlling cells' fate. Bcl-xL is one of anti-apoptotic protein in this family. In this study, a new Bcl-xL homolog (EcBcl-xL) was identified and characterized from orange-spotted grouper, Epinephelus coioides. EcBcl-xL encoded a 221 amino acid peptides that shared 86% identity to Larimichthys crocea Bcl-xL protein, contained four conserved BH domains and one transmembrane region. The predicted three-dimensional structure of EcBcl-xL was similar with Homo sapiens Bcl-xL. EcBcl-xL widely expressed in all tested tissues with highest expression in head kidney. Its expression level was significantly up-regulated after SGIV infection in vivo. Furthermore, overexpression of EcBcl-xL could inhibit SGIV-induced nonapoptotic cell death and suppressed viral genes transcriptions in GS cells. Our findings suggested that EcBcl-xL might play a role during virus infection through modulating SGIV-induced nonapoptotic cell death., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Viral and episomal DNAs, as signs of infections and dangers, induce a series of immune responses in the host, and cells must sense foreign DNAs to eliminate the invaders. The cell nucleus is not "immune privileged" and exerts intrinsic mechanisms to control nuclear-replicating DNA viruses. Thus, it is important to understand the action of viral DNA sensing in the cell nucleus. Here, we reveal a mechanism of restriction of DNA viruses and episomal plasmids mediated by PJA1, a RING-H2 E3 ubiquitin ligase. PJA1 restricts the DNA viruses hepatitis B virus (HBV) and herpes simplex virus 1 (HSV-1) but not the RNA viruses enterovirus 71 (EV71) and vesicular stomatitis virus (VSV). Similarly, PJA1 inhibits episomal plasmids but not chromosome-integrated reporters or endogenous genes. In addition, PJA1 has no effect on endogenous type I and II interferons (IFNs) and interferon-stimulated genes (ISGs), suggesting that PJA1 silences DNA viruses independent of the IFN pathways. Interestingly, PJA1 interacts with the SMC5/6 complex (a complex essential for chromosome maintenance and HBV restriction) to facilitate the binding of the complex to viral and episomal DNAs in the cell nucleus. Moreover, treatment with inhibitors of DNA topoisomerases (Tops) and knockdown of Tops release PJA1-mediated silencing of viral and extrachromosomal DNAs. Taken together, results of this work demonstrate that PJA1 interacts with SMC5/6 and facilitates the complex to bind and eliminate viral and episomal DNAs through DNA Tops and thus reveal a distinct mechanism underlying restriction of DNA viruses and foreign genes in the cell nucleus. IMPORTANCE DNA viruses, including hepatitis B virus and herpes simplex virus, induce a series of immune responses in the host and lead to human public health concerns worldwide. In addition to cytokines in the cytoplasm, restriction of viral DNA in the nucleus is an important approach of host immunity. However, the mechanism of foreign DNA recognition and restriction in the cell nucleus is largely unknown. This work demonstrates that an important cellular factor (PJA1) suppresses DNA viruses and transfected plasmids independent of type I and II interferon (IFN) pathways. Instead, PJA1 interacts with the chromosome maintenance complex (SMC5/6), facilitates the complex to recognize and bind viral and episomal DNAs, and recruits DNA topoisomerases to restrict the foreign molecules. These results reveal a distinct mechanism underlying the silencing of viral and episomal invaders in the cell nuclei and suggest that PJA1 acts as a potential agent to prevent infectious and inflammatory diseases., (Copyright © 2018 Xu et al.)

Intestine is a primary site of the white spot syndrome virus (WSSV) infection in most crustaceans. To date, little is known about its role in the anti-viral immune response in the freshwater prawn Macrobrachium rosenbergii. In this study, next-generation sequencing was employed to investigate the M. rosenbergii intestine transcriptomes following WSSV or poly I:C challenges. A total of 41.06 M, 39.58 M and 47.00 M clean reads were generated and assembled into 65,340, 71,241 and 70,614 transcripts from the negative control group (NG), WSSV challenge group (WG) and poly I:C treatment group (PG) respectively. Based on homology searches, functional annotation with 7 databases (NR, NT, GO, COG, KEGG, Swissprot and Interpro) for 88,412 transcripts was performed. After WSSV or poly (I:C) challenge, the numbers of up-regulated differentially expressed genes (DEGs) were greater than the down-regulated DEGs. Gene Ontology (GO) classification of the DEGs also distributed similarly, with the same top 10 annotations and were all assigned to the signaling pathways, including spliceosome, Rap1 signaling pathway, proteoglycans, PI3K-Akt signaling pathway, ECM receptor interaction. Results could contribute to a better understanding of the intestinal immune response to viral pathogens., Competing Interests: The authors have declared that no competing interests exist.

To assess the immune response of hemocytes to white spot syndrome virus (WSSV) or Vibrio alginolyticus infection in the mud crab Scylla paramamosain, a transcriptome analysis was performed. We report the analysis of 45131 transcripts from S. paramamosain hemocytes by de novo assembly. A comparison with GenBank protein and nucleotide sequences identified 33699 genes as previously known. The length distribution of the genes was 8147 genes ≥200 bp, 4714 genes ≥300 bp, and 3517 genes ≥2000 bp. A total of 21579 simple sequence repeats (SSRs) were found in the transcriptomic dataset, including 9% monomers, 53.34% dimers and 32.55% trimers. A total of 13172 and 5087 differentially expressed transcripts were found in the V. alginolyticus-infected group and WSSV-infected group, respectively. Of these, 5920 transcripts were up-regulated and 7252 were down-regulated in the V. alginolyticus-infected crabs and 2302 transcripts were up-regulated and 2785 were down-regulated in the WSSV-infected crabs. Additionally, 3096 transcripts were differentially expressed simultaneously in the V. alginolyticus-infected crabs and the WSSV-infected crabs. Several known immune-related genes such as heat shock protein, Janus kinase, STAT, relish, caspase, Ca 2+ -transporting ATPase and lysosomal alpha-mannosidase were found among the differentially expressed transcripts. Transcription and its regulation were significant biological processes, and ATP binding and zinc ion binding were significant molecular functions. This is the first report of comparative transcriptomic analysis of crab hemocytes in response to WSSV or V. alginolyticus infection. These findings will contribute to our understanding of the immune response to WSSV and V. alginolyticus infection in crustaceans., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Drosophila melanogaster has played a key role in our understanding of invertebrate immunity. However, both functional and evolutionary studies of host-virus interaction in Drosophila have been limited by a dearth of native virus isolates. In particular, despite a long history of virus research, DNA viruses of D. melanogaster have only recently been described, and none have been available for experimental study. Here we report the isolation and comprehensive characterisation of Kallithea virus, a large double-stranded DNA virus, and the first DNA virus to have been reported from wild populations of D. melanogaster. We find that Kallithea virus infection is costly for adult flies, reaching high titres in both sexes and disproportionately reducing survival in males, and movement and late fecundity in females. Using the Drosophila Genetic Reference Panel, we quantify host genetic variance for virus-induced mortality and viral titre and identify candidate host genes that may underlie this variation, including Cdc42-interacting protein 4. Using full transcriptome sequencing of infected males and females, we examine the transcriptional response of flies to Kallithea virus infection and describe differential regulation of virus-responsive genes. This work establishes Kallithea virus as a new tractable model to study the natural interaction between D. melanogaster and DNA viruses, and we hope it will serve as a basis for future studies of immune responses to DNA viruses in insects., Competing Interests: The authors have declared that no competing interests exist.

Humans have a close phylogenetic relationship with nonhuman primates (NHPs) and share many physiological parallels, such as highly similar immune systems, with them. Importantly, NHPs can be infected with many human or related simian viruses. In many cases, viruses replicate in the same cell types as in humans, and infections are often associated with the same pathologies. In addition, many reagents that are used to study the human immune response cross-react with NHP molecules. As such, NHPs are often used as models to study viral vaccine efficacy and antiviral therapeutic safety and efficacy and to understand aspects of viral pathogenesis. With several emerging viral infections becoming epidemic, NHPs are proving to be a very beneficial benchmark for investigating human viral infections.

Background: Torque teno virus is a small chronically persisting circular negative ssDNA virus reaching near 100% prevalence. It is reported to be a marker for immune function in immunocompromised patients. The possibility of vertical maternal-fetal transmission remains controversial but incidence rate of TTV DNA in children increased with age. TTV dynamics well studied for allogeneic hematopoietic stem cell transplantation as a predictor of post-transplant complications but there is no viral proliferation kinetics data for other patient groups or healthy individuals. The aim of this study was to determine TTV dynamics during the first year of life of healthy infants., Methods: Ninety eight clinically healthy breastfeeding infants (1-12 months of age) were analyzed by quantitative PCR for the whole blood TTV load with the test sensitivity of about 1000 viral copies per milliliter of blood (total number of samples including repeatedly tested infants was 109)., Results: 67% of all analyzed samples were TTV-positive demonstrating significant positive correlation between age and TTV load (r = 0.81, p < 0.01)., Conclusions: This is the first study to suggest that viral load increases during the first year of life reaching a plateau after 6 months with strong proliferation for the first 60 days. Our data well correlates with TTV dynamics in patients following allogeneic hematopoietic stem cell transplantation.

Ranaviruses are responsible for a lethal, emerging infectious disease in amphibians and threaten their populations throughout the world. Despite this, little is known about how amphibian populations respond to ranaviral infection. In the United Kingdom, ranaviruses impact the common frog (Rana temporaria). Extensive public engagement in the study of ranaviruses in the UK has led to the formation of a unique system of field sites containing frog populations of known ranaviral disease history. Within this unique natural field system, we used RNA sequencing (RNA-Seq) to compare the gene expression profiles of R. temporaria populations with a history of ranaviral disease and those without. We have applied a RNA read-filtering protocol that incorporates Bloom filters, previously used in clinical settings, to limit the potential for contamination that comes with the use of RNA-Seq in nonlaboratory systems. We have identified a suite of 407 transcripts that are differentially expressed between populations of different ranaviral disease history. This suite contains genes with functions related to immunity, development, protein transport and olfactory reception among others. A large proportion of potential noncoding RNA transcripts present in our differentially expressed set provide first evidence of a possible role for long noncoding RNA (lncRNA) in amphibian response to viruses. Our read-filtering approach also removed significantly more bacterial reads from libraries generated from positive disease history populations. Subsequent analysis revealed these bacterial read sets to represent distinct communities of bacterial species, which is suggestive of an interaction between ranavirus and the host microbiome in the wild., (© 2018 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.)

Lymphocystis disease virus (LCDV) infection may induce a variety of host gene expression changes associated with disease development; however, our understanding of the molecular mechanisms underlying host-virus interactions is limited. In this study, RNA sequencing (RNA-seq) was employed to investigate differentially expressed genes (DEGs) in the gill of the flounder ( Paralichthys olivaceus ) at one week post LCDV infection. Transcriptome sequencing of the gill with and without LCDV infection was performed using the Illumina HiSeq 2500 platform. In total, RNA-seq analysis generated 193,225,170 clean reads aligned with 106,293 unigenes. Among them, 1812 genes were up-regulated and 1626 genes were down-regulated after LCDV infection. The DEGs related to cellular process and metabolism occupied the dominant position involved in the LCDV infection. A further function analysis demonstrated that the genes related to inflammation, the ubiquitin-proteasome pathway, cell proliferation, apoptosis, tumor formation, and anti-viral defense showed a differential expression. Several DEGs including β actin , toll-like receptors, cytokine-related genes, antiviral related genes, and apoptosis related genes were involved in LCDV entry and immune response. In addition, RNA-seq data was validated by quantitative real-time PCR. For the first time, the comprehensive gene expression study provided valuable insights into the host-pathogen interaction between flounder and LCDV., Competing Interests: The authors declare no conflict of interest.

Members of the Iridoviridae family have been considered as aetiological agents of iridovirus diseases, causing fish mortalities and economic losses all over the world. Virus identification based on candidate gene sequencing is faster, more accurate and more reliable than other traditional phenotype methodologies. Iridoviridae viruses are covered by a protein shell (capsid) encoded by the important candidate gene, major capsid protein (MCP). In this study, we investigated the potential of the MCP gene for use in the diagnosis and identification of infections caused Megalocytivirus of the Iridoviridae family. We selected data of 66 Iridoviridae family isolates (53 strains of Megalocytivirus, eight strains of iridoviruses and five strains of Ranavirus) infecting various species of fish distributed all over the world. A total of 53 strains of Megalocytivirus were used for designing the complete primer sets for identifying the most hypervariable region of the MCP gene. Further, our in silico analysis of 102 sequences of related and unrelated viruses reconfirms that primer sets could identify strains more specifically and offers a useful and fast alternative for routine clinical laboratory testing. Our findings suggest that phenotype observation along with diagnosis using universal primer sets can help detect infection or carriers at an early stage., (© 2017 John Wiley & Sons Ltd.)

Due to high-density aquafarming in Taiwan, groupers are commonly infected with two different iridoviruses: Megalocytivirus (grouper iridovirus of Taiwan, TGIV) and Ranavirus (grouper iridovirus, GIV). Iridoviral diseases cause mass mortality, and surviving fish retain these pathogens, which can then be horizontally transferred. These viruses have therefore become a major challenge for grouper aquaculture. In this study, comparisons of the biological responses of groupers to infection with these two different iridoviruses were performed. A novel approach for transcriptomic analysis was proposed to enhance the discovery of differentially expressed genes and associated biological pathways. In this method, suitable and available reference species are selected from the NCBI taxonomy tree and the Ensembl and KEGG databases instead of either choosing only one model species or adopting the NCBI non-redundant dataset as references. Our results show that selection of multiple appropriate model species as references increases the efficiency and performance of analyses compared to those of traditional approaches. Using this method, 17 shared pathways and 5 specific pathways were found to be significantly differentially expressed following infection with the two iridoviruses, among which 11 pathways were additionally identified based on the proposed method of multiple reference species selection. Among the pathways responsive to infection with a specific iridovirus, the spliceosomal pathway (ko03040; p-value = 0.0011) was exclusively associated with TGIV infection, while the glycolysis/gluconeogenesis pathway (ko00010; p-value = 0.0032) was associated with GIV infection. These findings and designed corresponding biological experiments may facilitate a deeper understanding of the mechanisms by which both TGIV and GIV cause fatal infections, as well as the ways in which they induce different pathologies and symptoms. We believe that the proposed novel mechanism for de novo transcriptomic analysis provides superior and comprehensive functional annotations and that the resulting shared and specific pathways identified may help immunologists develop specific vaccines against various types of iridovirus in the near future., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Red sea bream iridoviral disease (RSIVD) is a major viral disease in red sea bream farming in Japan. Previously, we identified one candidate male individual of red sea bream that was significantly associated with convalescent individuals after RSIVD. The purpose of this study is to identify the quantitative trait loci (QTL) linked to the RSIVD-resistant trait for future marker-assisted selection (MAS). Two test families were developed using the candidate male in 2014 (Fam-2014) and 2015 (Fam-2015). These test families were challenged with RSIV, and phenotypes were evaluated. Then, de novo genome sequences of red sea bream were obtained through next-generation sequencing, and microsatellite markers were searched and selected for linkage map construction. One immune-related gene, MHC class IIβ, was also used for linkage map construction. Of the microsatellite markers searched, 148 and 197 were mapped on 23 and 27 linkage groups in the female and male linkage maps, respectively, covering approximately 65% of genomes in both sexes. One QTL linked to an RSIVD-resistant trait was found in linkage group 2 of the candidate male in Fam-2014, and the phenotypic variance of the QTL was 31.1%. The QTL was closely linked to MHC class IIβ. Moreover, the QTL observed in Fam-2014 was also significantly linked to an RSIVD-resistant trait in the candidate male of Fam-2015. Our results suggest that the RSIVD-resistant trait in the candidate male was controlled by one major QTL closely linked to the MHC class IIβ gene and could be useful for MAS of red sea bream.

Identifying quantitative trait loci (QTL) for viral disease resistance is of particular importance in selective breeding programs of fish species. Genetic markers linked to QTL can be useful in marker-assisted selection (MAS) for elites resistant to specific pathogens. Here, we conducted a genome scan for QTL associated with Singapore grouper iridovirus (SGIV) resistance in an Asian seabass (Lates calcarifer) family, using a high-density linkage map generated with genotyping-by-sequencing. One genome-wide significant and three suggestive QTL were detected at LG21, LG6, LG13, and LG15, respectively. The phenotypic variation explained (PVE) by the four QTL ranged from 7.5 to 15.6%. The position of the most significant QTL at LG21 was located between 31.88 and 36.81 cM. The SNP marker (SNP130416) nearest to the peak of this QTL was significantly associated with SGIV resistance in an unrelated multifamily population. One candidate gene, MECOM, close to the peak of this QTL region, was predicted. Evidence of alternative splicing was observed for MECOM and one specific category of splicing variants was differentially expressed at 5 days post-SGIV infection. The QTL detected in this study are valuable resources and can be used in the selective breeding programs of Asian seabass with regard to resistance to SGIV.

CD2 is expressed on the surfaces of virtually all T cells and natural killer (NK) cells. In mammals, the CD2 molecule is 50 kDa. The cytoplasmic tail of CD2 interacts with CD2-associated protein (CD2AP), which plays an important role in mediating the trigger signal in outer magnetic pole cells. In this study, we identified CD2AP from rock bream and investigated its gene expression. The ORF of CD2AP (1950 bp) encodes 650 amino acids (aa). CD2AP has a Src homology 3 (SH3) domain. Quantitative real-time PCR analysis revealed that CD2AP shows higher expression in the gills and skin. Under experimental challenge, CD2AP gene expression was increased as relative to the control after 7 days. This result will improve our understanding of blood vessels in teleost fish, and will provide a basis for the study of CD2-related genes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)