Haemolysins are essential to the pathogenicity of deep-sea Vibrio fluvialis.

Vibrio fluvialis is an emerging foodborne pathogen that produces VFH (Vibrio fluvialis hemolysin) and δVFH (delta-Vibrio fluvialis hemolysin). The function of δVFH is unclear. Currently, no pathogenic V. fluvialis from deep sea has been reported. In this work, a deep-sea V. fluvialis isolate (V13) was examined for pathogenicity. V13 was most closely related to V. fluvialis ATCC 33809, a human isolate, but possessed 262 unique genes. V13 caused lethal infection in fish and induced pyroptosis involving activation of the NLRP3 inflammasome, caspase 1 (Casp1), and gasdermin D (GSDMD). V13 defective in VFH or VFH plus δVFH exhibited significantly weakened cytotoxicity. Recombinant δVFH induced NLRP3-Casp1-GSDMD-mediated pyroptosis in a manner that depended on K+ efflux and intracellular Ca2+ accumulation. δVFH bound several plasma membrane lipids, and these bindings were crucial for δVFH cytotoxicity. Together these results provided new insights into the function of δVFH and the virulence mechanism of V. fluvialis.

Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner.

Bacillus cereus is a clinically significant foodborne pathogen that causes severe gastrointestinal and non-gastrointestinal disease. Cereolysin O (CLO) is a putative virulence factor of B. cereus, and its function remains to be investigated. In this study, we examined the biological activity of CLO from a deep sea B. cereus isolate. CLO was highly toxic to mammalian cells and triggered pyroptosis through NLRP3 inflammasome-mediated caspase 1 and gasdermin D activation. CLO-induced cell death involved ROS accumulation and K+ efflux, and was blocked by serum lipids. CLO bound specifically to cholesterol, and this binding was essential to CLO cytotoxicity. The structural integrity of the three tryptophan residues in the C-terminal undecapeptide was vital for CLO to interact with membrane lipids and cause membrane perforation. Taken together, these results provided new insights into the molecular mechanism of B. cereus CLO-mediated cytotoxicity.

A novel teleost microRNA regulates autophagy and NF-κB activation during bacterial infection.

MicroRNAs (miRNAs) are a class of non-coding RNAs with regulatory functions in many cellular processes, including immune defense. In this study, we identified novel-m0089-3p, a novel miRNA with unknown function, in the teleost fish Japanese flounder (Paralichthys olivaceus) and investigated its immune function. Novel-m0089-3p was found to target the autophagy-associated gene ATG7 and negatively regulate ATG7 expression via interaction with the 3' UTR of ATG7. During the infection of the bacterial pathogen Edwardsiella tarda, novel-m0089-3p expression was induced in flounder, which in turn repressed ATG7 expression. Overexpression of novel-m0089-3p or blocking ATG7 expression inhibited autophagy and promoted the intracellular replication of E. tarda. Novel-m0089-3p overexpression, as well as E. tarda infection, activated NF-κB and stimulated the expression of inflammatory cytokines. Together these results revealed an important role of novel-m0089-3p in response to bacterial infection.

Japanese Flounder pol-miR-155 Is Involved in Edwardsiella tarda Infection via ATG3.

MicroRNAs (miRNAs) are small RNA molecules that function in the post-transcriptionally regulation of the expression of diverse genes, including those involved in immune defense. Edwardsiella tarda can infect a broad range of hosts and cause severe disease in aquatic species, including Japanese flounder (Paralichthys olivaceus). In this study, we examined the regulation mechanism of a flounder miRNA, pol-miR-155, during the infection of E. tarda. Pol-miR-155 was identified to target flounder ATG3. Overexpression of pol-miR-155 or knockdown of ATG3 expression suppressed autophagy and promoted the intracellular replication of E. tarda in flounder cells. Overexpression of pol-miR-155 activated the NF-κB signaling pathway and further promoted the expression of downstream immune related genes of interleukin (IL)-6 and IL-8. These results unraveled the regulatory effect of pol-miR-155 in autophagy and in E. tarda infection.

A saposin domain-containing protein of tongue sole Cynoglossus semilaevis: Antimicrobial activity and mechanism.

Prosaposin is a precursor that can be processed into four different saposins, designated as A, B, C, and D, which have multiple functions in mammals, including neuroprotection and immune modulation. The immune function of saposin in teleost remains largely unknown. In the present study, a saposin (SAP) domain-containing protein was identified in half-smooth tongue sole Cynoglossus semilaevis and named CsSDP. CsSDP harbors one SAP A domain and two SAP B domains. When expressed in HEK293T cells, CsSDP was specifically localized in the lysosome. When overexpressed in Escherichia coli, CsSDP markedly inhibited bacterial growth, and the inhibitory effect depended on two specific regions in the SAP A and SAP B domains. Two polypeptides (P32 and P30) derived from the above SAP A and B domains could bind to and inhibit the growth of both Gram-negative and Gram-positive bacteria. The ultrastructural analysis revealed that P32 and P30 killed target bacteria by disrupting the bacterial cell wall and inducing substantial release of cytoplasmic contents. These results shed new lights on the immune function of saposin domain-containing protein in teleost.

TNFR2 is a regulatory target of pol-miR-194a and promotes the antibacterial immunity of Japanese flounder Paralichthys olivaceus.

MicroRNAs (miRNAs) are regulatory RNAs that modulate target gene expression after transcription. Pol-miR-194a had been reported to be a miRNA of Japanese flounder (Paralichthys olivaceus) involved in Edwardsiella tarda infection. Here, we identified tumor necrosis factor receptor 2 (TNFR2) as a target gene of pol-miR-194a. Pol-miR-194a markedly repressed the protein expression of flounder TNFR2 (PoTNFR2) via specific interaction with the 3'UTR of PoTNFR2. PoTNFR2 responded to E. tarda infection in a manner that was opposite to that of pol-miR-194a and inhibited E. tarda invasion by activating the NF-κB pathway. Consistently, dysregulation of PoTNFR2 had a significant impact on E. tarda dissemination in flounder tissues. Together, these results add new insights into the regulation mechanism and immune function of fish TNFR2 and pol-miR-194a.

A Novel microRNA of Japanese Flounder Regulates Antimicrobial Immunity Involving a Bacteria-Binding CSF3.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate diverse biological processes including immunity. In a previous high-throughput RNA sequencing study, a novel miRNA, pol-miR-novel_642, was identified from Japanese flounder (Paralichthys olivaceus), a farmed fish species with important economic value. In this study, we investigated the regulatory mechanism and the function of pol-miR-novel_642 and its target gene. We found that pol-miR-novel_642 targeted, in a sequence-specific manner, a flounder gene encoding an uncharacterized protein that is a structural homologue of murine granulocyte colony stimulating factor 3 (CSF3). The expression of pol-miR-novel_642 and its target gene (named PoCSF3-1) was regulated, in different manners, by the bacterial pathogen Edwardsiella tarda and the viral pathogen megalocytivirus. Overexpression of pol-miR-novel_642 or interference with PoCSF3-1 expression in flounder cells strongly potentiated E. tarda infection. Consistently, in vivo knockdown of PoCSF3-1 enhanced bacterial dissemination in flounder tissues but blocked viral replication, whereas in vivo overexpression of PoCSF3-1 inhibited bacterial dissemination and facilitated viral infection. Overexpression/knockdown of PoCSF3-1 and pol-miR-novel_642 also affected the activation of autophagy. Recombinant PoCSF3-1 (rPoCSF3-1) interacted with and inhibited the growth of Gram-negative bacteria in a manner relying on a PoCSF3-1-characteristic structural motif that is absent in mouse CSF3. rPoCSF3-1 also regulated the proliferation, inflammatory response, and immune defense of flounder head kidney leukocytes in a structure-dependent fashion. Together, these results reveal the function of a novel miRNA-CSF3 regulatory system of flounder, and add new insights into the role and mechanism of fish miRNA and CSF3 in antimicrobial immunity.

PUF60 of Japanese flounder is regulated by pol-miR-novel_395 and involved in pathogen infection, autophagy, and apoptosis.

MicroRNAs (miRNAs) are evolutionary conserved, non-coding small RNAs that have been shown to regulate diverse biological processes including immunity. In a previous study, a novel miRNA of Japanese flounder (Paralichthys olivaceus), pol-miR-novel_395, was found to be responsive in expression to the infection of the bacterial pathogen Edwardsiella tarda. In the present study, we examined the regulation and immune effect of pol-miR-novel_395 and its target gene. We found that pol-miR-novel_395 expression was regulated by E. tarda and megalocytivirus, and pol-miR-novel_395 targeted the gene of PUF60 (poly (U)-binding-splicing factor 60 kDa) of flounder (named PoPUF60). Constitutive expression of PoPUF60 occurred in relatively high levels in the heart and liver of flounder. Bacterial infection upregulated PoPUF60 expression, whereas viral infection downregulated PoPUF60 expression. Interference with PoPUF60 expression or overexpression of pol-miR-novel_395 in flounder cells strongly potentiated E. tarda infection. Consistently, in vivo knockdown of PoPUF60 enhanced bacterial dissemination in the tissues of flounder but blocked viral replication, whereas in vivo overexpression of PoPUF60 inhibited bacterial dissemination but facilitated viral replication. Additionally, pol-miR-novel_395 and PoPUF60 were involved in the process of autophagy and apoptosis. Collectively, these results indicated that PoPUF60 and pol-miR-novel_395 play an important role in pathogen infection, autophagy, and apoptosis.

A virulent Bacillus cereus strain from deep-sea cold seep induces pyroptosis in a manner that involves NLRP3 inflammasome, JNK pathway, and lysosomal rupture.

Recent studies indicate that the Bacillus species is distributed in deep-sea environments. However, no specific studies on deep-sea Bacillus cereus have been documented. In the present work, we isolated a B. cereus strain, H2, from the deep-sea cold seep in South China Sea. We characterized the pathogenic potential of H2 and investigated H2-induced death of different types of cells. We found that H2 was capable of tissue dissemination and causing acute mortality in mice and fish following intraperitoneal/intramuscular injection. In vitro studies revealed that H2 infection of macrophages induced pyroptosis and activation of the NLRP3 inflammasome pathway that contributed partly to cell death. H2 infection activated p38, JNK, and ERK, but only JNK proved to participate in H2-triggered cell death. Reactive oxygen species (ROS) and intracellular Ca2+ were essential to H2-induced activation of JNK and NLRP3 inflammasome. In contrast, lysosomal rupture and cathepsins were required for H2-induced NLRP3 inflammasome activation but not for JNK activation. This study revealed for the first time the virulence characteristics of deep-sea B. cereus and provided new insights into the mechanism of B. cereus infection.

Phosphatase and Tensin Homolog (PTEN) of Japanese Flounder-Its Regulation by miRNA and Role in Autophagy, Apoptosis and Pathogen Infection.

MicroRNAs (miRNAs) are small non-coding RNAs with important roles in diverse biological processes including immunity. Japanese flounder (Paralichthys olivaceus) is an aquaculture fish species susceptible to the infection of bacterial and viral pathogens including Edwardsiella tarda. In a previous study, pol-miR-novel_547, a novel miRNA of flounder with unknown function, was found to be induced by E. tarda. In the present study, we investigated the regulation and function of pol-miR-novel_547 and its target gene. We found that pol-miR-novel_547 was regulated differently by E. tarda and the viral pathogen megalocytivirus, and pol-miR-novel_547 repressed the expression of PTEN (phosphatase and tensin homolog) of flounder (PoPTEN). PoPTEN is ubiquitously expressed in multiple tissues of flounder and responded to bacterial and viral infections. Interference with PoPTEN expression in flounder cells directly or via pol-miR-novel_547 promoted E. tarda invasion. Consistently, in vivo knockdown of PoPTEN enhanced E. tarda dissemination in flounder tissues, whereas in vivo overexpression of PoPTEN attenuated E. tarda dissemination but facilitated megalocytivirus replication. Further in vitro and in vivo studies showed that PoPTEN affected autophagy activation via the AKT/mTOR pathway and also modulated the process of apoptosis. Together these results reveal for the first time a critical role of fish PTEN and its regulatory miRNA in pathogen infection, autophagy, and apoptosis.

The Bipartite Sequence Motif in the N and C Termini of gp85 of Subgroup J Avian Leukosis Virus Plays a Crucial Role in Receptor Binding and Viral Entry.

Subgroup J avian leukemia virus (ALV-J), belonging to the genus Alpharetrovirus, enters cells through its envelope surface unit (gp85) via specifically recognizing the cellular receptor chicken Na+/H+ exchanger type I (chNHE1), the 28 to 39 N-terminal residues of which were characterized as the minimal receptor functional domain in our previous studies. In this study, to further clarify the precise organization and properties of the interaction between ALV-J gp85 and chNHE1, we identified the chNHE1-binding domain of ALV-J gp85 using a series of gp85 mutants with segment substitutions and evaluating their effects on chNHE1 binding in protein-cell binding assays. Our results showed that hemagglutinin (HA) substitutions of amino acids (aa) 38 to 131 (N terminus of gp85) and aa 159 to 283 (C terminus of gp85) significantly inhibited the interaction between gp85 and chNHE1/chNHE1 loop 1. In addition, these HA-substituted chimeric gp85 proteins could not effectively block the entry of ALV-J into chNHE1-expressing cells. Furthermore, analysis of various N-linked glycosylation sites and cysteine mutants in gp85 revealed that glycosylation sites (N6 and N11) and cysteines (C3 and C9) were directly involved in receptor-gp85 binding and important for the entry of ALV-J into cells. Taken together, our findings indicated that the bipartite sequence motif, spanning aa 38 to 131 and aa 159 to 283, of ALV-J gp85 was essential for binding to chNHE1, with its two N-linked glycosylation sites and two cysteines being important for its receptor-binding function and subsequent viral infection steps.IMPORTANCE Infection of a cell by retroviruses requires the attachment and fusion of the host and viral membranes. The specific adsorption of envelope (Env) surface proteins to cell receptors is a key step in triggering infections and has been the target of antiviral drug screening. ALV-J is an economically important avian pathogen that belongs to the genus Alpharetrovirus and has a wider host range than other ALV subgroups. Our results showed that the amino acids 38 to 131 of the N terminus and 159 to 283 of the C terminus of ALV-J gp85 controlled the efficiency of gp85 binding to chNHE1 and were critical for viral infection. In addition, the glycosylation sites (N6 and N11) and cysteines (C3 and C9) of gp85 played a crucial role in the receptor binding and viral entry. These findings might help elucidate the mechanism of the entry of ALV-J into host cells and provide antiviral targets for the control of ALV-J.

Pol-miR-363-3p plays a significant role in the immune defense of Japanese flounder Paralichthys olivaceus against bacterial and viral infection.

In this study, we examined the function of a Japanese flounder (Paralichthys olivaceus) microRNA (miRNA), pol-miR-363-3p. We found that pol-miR-363-3p targets an ubiquitin-specific protease (USP), USP32. USP is a family of deubiquitinating enzymes essential to the functioning of the ubiquitin proteasome system. In mammals, USP32 is known to be associated with cancer and immunity. In fish, the function of USP32 is unknown. We found that flounder USP32 (PoUSP32) expression was detected in the major tissues of flounder, particularly intestine. In vitro and in vivo studies showed that pol-miR-363-3p directly regulated PoUSP32 in a negative manner by interaction with the 3'UTR of PoUSP32. Overexpression of pol-miR-363-3p or interference with PoUSP32 expression in flounder cells significantly blocked Streptococcus iniae infection. Consistently, in vivo knockdown of pol-miR-363-3p or overexpression of PoUSP32 enhanced dissemination of S. iniae in flounder tissues, whereas in vivo knockdown of PoUSP32 inhibited S. iniae dissemination. In addition, pol-miR-363-3p knockdown also significantly promoted the tissue dissemination of the viral pathogen megalocytivirus, which, as well as S. iniae, regulated pol-miR-363-3p expression. Together these results revealed an important role of pol-miR-363-3p in flounder immune defense against bacterial and viral infection.

The novel fish miRNA pol-miR-novel_171 and its target gene FAM49B play a critical role in apoptosis and bacterial infection.

MicroRNAs (miRNAs) are a type of small, non-coding RNAs that participate in many cellular and biological processes by regulating mRNA stability. In a previous study, we identified 96 Japanese flounder (Paralichthys olivaceus) miRNAs responsive to the infection of Edwardsiella tarda, a bacterial pathogen to fish as well as humans. In the current study, we examined the regulation and function of one novel miRNA, i.e., pol-miR-novel_171, from the above 96 miRNA pool. We found that pol-miR-novel_171 expression was regulated by E. tarda and megalocytivirus in a pathogen-specific manner, and that pol-miR-novel_171 targeted the gene of FAM49B (family with sequence similarity 49 member B) of flounder (named PoFAM49B) by negative interaction with the 3'-UTR of PoFAM49B. To date, the function fish FAM49B is unknown. We found that PoFAM49B expressed in multiple tissues of flounder, and recombinant PoFAM49B interacted with and inhibited the growth of Gram-negative bacterial pathogens. Interference with PoFAM49B expression in flounder cells promoted E. tarda infection. Similar effects on E. tarda infection were observed with pol-miR-novel_171 overexpression. Consistently, in vivo knockdown of PoFAM49B in flounder enhanced E. tarda dissemination in fish tissues. Furthermore, interference with PoFAM49B expression, or overexpression of pol-miR-novel_171, promoted apoptosis of flounder cells, while in vitro and in vivo knockdown of PoFAM49B augmented the expressions of key apoptosis-associated genes. These results revealed for the first time the immune function of fish FAM49B and the regulatory mechanism of a novel fish miRNA by demonstrating that pol-miR-novel_171, via PoFAM49B, played a critical role in apoptosis and anti-bacterial immunity.

Japanese flounder pol-miR-3p-2 suppresses Edwardsiella tarda infection by regulation of autophagy via p53.

MicroRNAs (miRNAs) are post-transcriptional regulators that play vital roles in diverse physiological processes including immunity. In this study, we investigated the regulatory mechanism and function of a novel Japanese flounder (Paralichthys olivaceus) miRNA, pol-miR-3p-2. pol-miR-3p-2 was responsive in expression to the infection of the bacterial pathogen Edwardsiella tarda. pol-miR-3p-2 negatively regulated the expression of p53 through interaction with the 3'UTR of p53. Overexpression of pol-miR-3p-2 promoted autophagy, resulting in augmented production of LC3-II, while knockdown of p53 increased the level of beclin, a key factor of autophagy. In vivo and in vitro studies showed that E. tarda infection induced autophagy in flounder, and pol-miR-3p-2 inhibited the infectivity of E. tarda. Together these results indicate that pol-miR-3p-2 regulates autophagy through the target gene p53, thus revealing a regulatory link between p53 and autophagy in teleost, and that pol-miR-3p-2 plays an important role in the immune defense against E. tarda.

Characterization of Streptococcus iniae-induced microRNA profiles in Paralichthys olivaceus and identification of pol-3p-10740_175 as a regulator of antibacterial immune response.

MicroRNAs (miRNAs) are involved in many biological activities including immune defense against pathogens. In this study, we applied high-throughput sequencing technology to examine miRNAs in Japanese flounder (Paralichthys olivaceus) infected with Streptococcus iniae at different times. A total of 1038 miRNAs were identified, of which, 249 were novel miRNAs, and 81 showed differential expression (named DEmiRNAs) after S. iniae infection. Of the 81 DEmiRNAs identified, 34 and 58 occurred at 6 h and 24 h post-infection, respectively; most DEmiRNAs were strongly time-specific, and only 13.6% of the DEmiRNAs were shared between the two time points. A total of 9582 target genes were predicted for the 81 DEmiRNAs. The putative target genes were enriched in various GO and KEGG pathways of biological processes and molecular/cellular functions, in particular endocytosis, regulation of transcription, lysososme, and the signaling pathways of MAPK, ErbB, and AMPK. One of the DEmiRNAs, pol-3p-10740_175, was found to target dual specificity phosphatase 6 (Dusp6) and repress the expression of the latter. Transfection of flounder FG cells with pol-3p-10740_175 caused a significant inhibition on S. iniae invasion. The results of this study provided the first S. iniae-induced miRNA profile in Japanese flounder and indicated that flounder miRNAs play an important role in antibacterial immunity.

pol-miR-194a of Japanese flounder (Paralichthys olivaceus) suppresses type I interferon response and facilitates Edwardsiella tarda infection.

MicroRNAs (miRNAs) are a type of small non-coding RNAs that participate in diverse cellular processes including microbial invasion and immune defense. In a previous study, we identified a large amount of Japanese flounder (Paralichthys olivaceus) miRNAs responsive to megalocytivirus infection. In the present study, we examined the function of one of these miRNAs, pol-miR-194a, in association with the infectivity of Edwardsiella tarda, an intracellular bacterial pathogen to many fish species including flounder. We found that pol-miR-194a was induced in expression to a significant extent in the spleen, liver, and gill of Japanese flounder infected by E. tarda. Transfection of flounder cells with pol-miR-194a mimic significantly enhanced the intracellular replication of E. tarda. pol-miR-194a was able to interact specifically with the 3'UTR of IRF7 in a negative manner, resulting in inhibition of IRF7 expression. Consistently, pol-miR-194a significantly blocked the promoter activity of type Ⅰ interferon. Taken together, these results indicate that pol-miR-194a plays an important role in the regulation of flounder immune response as well as microbial infection, and that pol-miR-194a probably serves as a target for E. tarda to manipulate and escape host immune defense.

The high conserved cellular receptors of avian leukosis virus subgroup J in Chinese local chickens contributes to its wide host range.

Avian leukosis virus (ALV) is a tumor-inducing virus that spreads among most chicken species, causing serious financial losses for the poultry industry. Subgroup J avian leukosis virus (ALV-J) is a recombinant exogenous ALV, which shows more extensive host range in comparison with other subgroups, especially in Chinese local chickens. To identify the relationship between ALV-J host range and the polymorphism of its cellular receptors, we performed a wide range epidemiological investigation of current ALV-J infection in Chinese local chickens, and discovered that all the 18 local chicken breeds being investigated from main local chicken breeding provinces were ALV-J positive. Furthermore, we cloned ALV-J cellular receptor genes of chNHE1 and chANXA2 of these 18 chicken breeds. Sequence alignment demonstrated that despite several regular mutations at the nucleotide level, there were no corresponding amino acid mutations for either chNHE1 gene or chANXA2 gene. Additionally, virus entry assay indicated that the level of viral enter into cells is stable among different chicken breeds. Results of this study indicated that the wide host range of ALV-J in Chinese local chickens was partially due to the high conservatism of its cellular receptors, and also provide target sites for drug design of resistance to ALV-J infection.

Residues 28 to 39 of the Extracellular Loop 1 of Chicken Na+/H+ Exchanger Type I Mediate Cell Binding and Entry of Subgroup J Avian Leukosis Virus.

Chicken Na+/H+ exchanger type I (chNHE1), a multispan transmembrane protein, is a cellular receptor of the subgroup J avian leukosis virus (ALV-J). To identify the functional determinants of chNHE1 responsible for the ALV-J receptor activity, a series of chimeric receptors was created by exchanging the extracellular loops (ECL) of human NHE1 (huNHE1) and chNHE1 and by ECL replacement with a hemagglutinin (HA) tag. These chimeric receptors then were used in binding and entry assays to map the minimal ALV-J gp85-binding domain of chNHE1. We show that ECL1 of chNHE1 (chECL1) is the critical functional ECL that interacts directly with ALV-J gp85; ECL3 is also involved in ALV-J gp85 binding. Amino acid residues 28 to 39 of the N-terminal membrane-proximal region of chECL1 constitute the minimal domain required for chNHE1 binding of ALV-J gp85. These residues are sufficient to mediate viral entry into ALV-J nonpermissive cells. Point mutation analysis revealed that A30, V33, W38, and E39 of chECL1 are the key residues mediating the binding between chNHE1 and ALV-J gp85. Further, the replacement of residues 28 to 39 of huNHE1 with the corresponding chNHE1 residues converted the nonfunctional ALV-J receptor huNHE1 to a functional one. Importantly, soluble chECL1 and huECL1 harboring chNHE1 residues 28 to 39 both could effectively block ALV-J infection. Collectively, our findings indicate that residues 28 to 39 of chNHE1 constitute a domain that is critical for receptor function and mediate ALV-J entry.IMPORTANCE chNHE1 is a cellular receptor of ALV-J, a retrovirus that causes infections in chickens and serious economic losses in the poultry industry. Until now, the domains determining the chNHE1 receptor function remained unknown. We demonstrate that chECL1 is critical for receptor function, with residues 28 to 39 constituting the minimal functional domain responsible for chNHE1 binding of ALV-J gp85 and efficiently mediating ALV-J cell entry. These residues are located in the membrane-proximal region of the N terminus of chECL1, suggesting that the binding site of ALV-J gp85 on chNHE1 is probably located on the apex of the molecule; the receptor-binding mode might be different from that of retroviruses. We also found that soluble chECL1, as well as huECL1 harboring chNHE1 residues 28 to 39, effectively blocked ALV-J infection. These findings contribute to a better understanding of the ALV-J infection mechanism and also provide new insights into the control strategies for ALV-J infection.

Avian leukosis virus subgroup J induces VEGF expression via NF-κB/PI3K-dependent IL-6 production.

Avian leukosis virus subgroup J (ALV-J) is an oncogenic virus causing hemangiomas and myeloid tumors in chickens. Interleukin-6 (IL-6) is a multifunctional pro-inflammatory interleukin involved in many types of cancer. We previously demonstrated that IL-6 expression was induced following ALV-J infection in chickens. The aim of this study is to characterize the mechanism by which ALV-J induces IL-6 expression, and the role of IL-6 in tumor development. Our results demonstrate that ALV-J infection increases IL-6 expression in chicken splenocytes, peripheral blood lymphocytes, and vascular endothelial cells. IL-6 production is induced by the ALV-J envelope protein gp85 and capsid protein p27 via PI3K- and NF-κB-mediated signaling. IL-6 in turn induced expression of vascular endothelial growth factor (VEGF)-A and its receptor, VEGFR-2, in vascular endothelial cells and embryonic vascular tissues. Suppression of IL-6 using siRNA inhibited the ALV-J induced VEGF-A and VEGFR-2 expression in vascular endothelial cells, indicating that the ALV-J-induced VEGF-A/VEGFR-2 expression is mediated by IL-6. As VEGF-A and VEGFR-2 are important factors in oncogenesis, our findings suggest that ALV-J hijacks IL-6 to promote tumorigenesis, and indicate that IL-6 could potentially serve as a therapeutic target in ALV-J infections.