Role of small nucleolar RNAs in alternative splicing of the doublesex gene in the silkworm, Bombyx mori.

More and more evidence shows that small noncoding RNAs (ncRNAs) play diverse roles in development, stress response and other cellular processes, but functional study of intermediate-size ncRNAs is still rare. Here, the expression profile of 16 intermediate-size ncRNAs in ovary and testis of silkworm Bombyx mori were analyzed. Twelve ncRNAs, including 5 small nucleolar RNAs (snoRNAs) and 7 unclassified ncRNAs, accumulated more in the testis than in the ovary of silkworm, especially Bm-163, Bm-51 and Bm-68. Four ncRNAs (including three orphan snoRNAs and one unclassified ncRNA) had higher expression level in the ovary than in the testis, especially Bm-86. Overexpression of the testis-enriched snoRNA Bm-68 in the female led to the accumulation of male-specific isoform of doublesex (BmdsxM) and increased the expression ratio of BmdsxM: BmdsxF. While overexpression of ovary-enriched snoRNA Bm-86 in the male decreased the expression ratio of BmdsxM: BmdsxF, indicating the roles of the two snoRNAs played in the alternative splicing of Bmdsx of silkworm, which will provide new clues for the functional study of snoRNAs in insects.

Red-grouper nervous necrosis virus B1 protein inhibits fish IFN response by targeting Ser5-phosphorylated RNA polymerase II to promote viral replication.

Nervous necrosis virus (NNV) could infect more than 200 fish species worldwide, with almost 100% mortality in affected larvae and juvenile fish. Among different genotypes of NNV, the red-grouper nervous necrosis virus (RGNNV) genotype is the most widely reported with the highest number of susceptible species. Interferon (IFN) is a crucial antiviral cytokine and RGNNV needs to develop some efficient strategies to resist host IFN-stimulated antiviral immune. Although considerable researches on RGNNV, whether RGNNV B1 protein participates in regulating the host's IFN response remains unknown. Here, we reported that B1 protein acted as a transcript inhibition factor to suppress fish IFN production. We firstly found that ectopic expression of B1 protein significantly decreased IFN and IFN-stimulated genes (ISGs) mRNA levels and IFNφ1 promoter activity induced by polyinosinic:polycytidylic acid [poly (I:C)]. Further studies showed that B1 protein inhibited the IFNφ1 promoter activity stimulated by the key RIG-I-like receptors (RLRs) factors, including MDA5, MAVS, TBK1, IRF3, and IRF7 and decreased their protein levels. Moreover, B1 protein significantly inhibited the activity of constitutively active cytomegalovirus (CMV) promoter, which suggested that B1 protein was a transcription inhibitor. Western blot indicated that B1 protein decreased the Ser5 phosphorylation of RNA polymerase II (RNAP II) C-terminal domain (CTD). Together, our data demonstrated that RGNNV B1 protein was a host transcript antagonist, which intervened RNAP II Ser5-phosphorylation, inhibiting host IFN response and facilitating RGNNV replication.

Molecular cloning, expression and functional analysis of STAT2 in orange-spotted grouper, Epinephelus coioides.

Signal transducer and activator of transcription 2 (STAT2) is an important molecule involved in the type I interferon signaling pathway. To better understand the functions of STAT2 in fish immune response, a STAT2 gene from orange-spotted grouper (Epinephelus coioides) (EcSTAT2) was cloned and characterized in this study. EcSTAT2 encoded a 802-amino acid peptide which shared 99.5% and 91.5% identity with giant grouper (Epinephelus lanceolatus) and leopard coral grouper (Plectropomus leopardus), respectively. Amino acid alignment analysis showed that EcSTAT2 contained five conserved domains, including N-terminal protein interaction domain, coiled coil domain (CCD), DNA binding domain (DBD), Src-homology 2 (SH2) domain, and C-terminal transactivation domain (TAD). Phylogenetic analysis indicated that EcSTAT2 clustered into fish STAT2 group and showed the nearest relationship to giant grouper STAT2. In healthy grouper, EcSTAT2 was distributed in all tissues tested, and the expression of EcSTAT2 was predominantly detected in spleen, kidney and gill. In vitro, EcSTAT2 expression was significantly increased in response to polyinosinic:polycytidylic acid [poly (I:C)] stimulation and red-spotted grouper nervous necrosis virus (RGNNV) infection. Subcellular localization showed that EcSTAT2 was located in the cytoplasm in a punctate manner. EcSTAT2 overexpression significantly inhibited RGNNV replication, as evidenced by the decreased severity of cytopathic effect (CPE) and the reduced expression levels of viral genes and protein. Consistently, knockdown of EcSTAT2 using small interfering RNA (siRNA) promoted RGNNV replication. Furthermore, EcSTAT2 overexpression increased both interferon (IFN) and interferon stimulated genes (ISGs) expression. In addition, EcSTAT2 knockdown decreased the transcription levels of IFN and ISGs. Together, our data demonstrated that EcSTAT2 exerted antiviral activity against RGNNV through up-regulation of host interferon response.

A pH-Driven Small-Molecule Nanotransformer Hijacks Lysosomes and Overcomes Autophagy-Induced Resistance in Cancer.

Smart conversion of supramolecular structures in vivo is an attractive strategy in cancer nanomedicine, which is usually achieved via specific peptide sequences. Here we developed a lysosomal targeting small-molecule conjugate, PBC, which self-assembles into nanoparticles at physiological pH and smartly converts to nanofibrils in lysosomes of tumor cells. Such a transformation mechanically leads to lysosomal dysfunction, autophagy inhibition, and unusual cytoplasmic vacuolation, thus granting PBC a unique anticancer activity as a monotherapy. Importantly, the photo-activated PBC elicits significant phototoxicity to lysosomes and shows enormous advantages in overcoming autophagy-caused treatment resistance frequently occurring in conventional phototherapy. This improved phototherapy achieves a complete cure of oral cancer xenografts upon limited administration. Our work provides a new paradigm for the construction of nonpeptide nanotransformers with biomedical activities.

Characterization of TCR+ and CD8+ head kidney leucocytes in Japanese flounder (Paralichthys olivaceus) with antisera against TCRα and CD8α.

T lymphocytes play an important role in cellular and adaptive immunity in vertebrates. The mechanisms of the fish immune system are little studied because of the lack of population-specific antibodies. This study examined the expression of two T lymphocyte markers, TCRα (PoTCRα) and CD8α (PoCD8α) in the Japanese flounder (Paralichthys olivaceus). The expression of PoTCRα and PoCD8α was mainly detected in immune/mucosal tissues. Recombinant PoTCRα and PoCD8α were expressed in pET32a and pET259, respectively. Then, rabbit anti-PoTCRα serum and rat anti-PoCD8α serum were prepared. Using serum, the characteristics of TCR+ and CD8+ head kidney leucocytes (HKLs) were investigated. The results of laser scanning confocal microscopy (LSCM) demonstrated that TCRα and CD8α were transmembrane proteins localized on the cell surface. The populations of CD8α- , CD8α+ , TCRα- , and TCRα+ were sorted by flow cytometry (FCM) and analysed using qRT-PCR. The results demonstrated that all TCRα+ /TCRα- or CD8α+ /CD8α- HKLs expressed IFN-γ. The CD4-1 and IgM transcripts were detected only in TCRα- and CD8α- cells. Furthermore, HKL mitogenesis was induced with concanavalin A (ConA) stimulation. Taken together, the results from LSCM and FCM analyses showed that mammalian and P. olivaceus TCR+ and CD8+ leucocytes share basic characteristics.

Characterization of two newly emerged torque teno sus virus isolates from a large-scale pig farm in China, in 2018.

Torque teno sus virus (TTSuV) infection is common in China's pig herd. Although of uncertain pathogenicity, TTSuVs have been reported as a worsening factor of other porcine diseases, including porcine circovirus associated disease (PCVAD), porcine respiratory diseases complex (PRDC) or porcine dermatitis and nephropathy syndrome (PDNS). To better understand the genetic diversity in TTSuVs, the complete genomes of two newly emerged isolates, referred to as HeN1-A9 and HeN1-A11, collected from pig samples at a large-scale pig farm in China, were analyzed. Phylogenetic relationships of TTSuV sequences separated TTSuV1 and TTSuVk2a groups and divided TTSuV1 into two major subtypes, including TTSuV1a and TTSuV1b; HeN1-A9 and HeN1-A11 strains classified into the TTSuV1a subtype. Recombination analysis demonstrated HeN1-A9 and HeN1-A11 were generated via recombination in the overlapping ORF1/ORF3 region of TTSuV1a genome, which we report for the first time. Furthermore, we found that HeN1-A9 could be replicated in cultured MARC-145 cells for 18 passages. Our findings may be useful for elucidating the characteristics and epidemic status of TTSuVs in China.

Molecular characterization and antibacterial immunity functional analysis of liver-expressed antimicrobial peptide 2 (LEAP-2) gene in golden pompano (Trachinotus ovatus).

Liver-expressed antimicrobial peptide-2 (LEAP-2) is a member of the antimicrobial peptides family. Research has demonstrated that LEAP-2 contains a number of cations and plays a key role in the innate immune system of organism. In this study, we cloned and identified TroLEAP-2, from the golden pompano (Trachinotus ovatus), and analyzed its functions in vivo and in vitro. Results showed that TroLEAP-2 contains a 321 bp open reading frame (ORF) that encodes 106 putative amino acids with a molecular weight of 11.65 kDa. The mature TroLEAP-2 peptide possesses four conserved cysteine residues, which can form a core structure with two disulfide bonds between the cysteine residues in the relative 1-3 (Cys 77 and Cys 88) and 2-4 (Cys 83 and Cys 93) positions. It has a high amino acid sequence similarity (38.68%-83.02%) with the liver-expressed antimicrobial peptide -2 of other teleosts. Phylogenetic analysis showed that TroLEAP-2 clustered with the LEAP-2 of Paralichthys olivaceus and Miichthy milluy. TroLEAP-2 was most abundantly expressed in the liver, spleen, and kidney, and was significantly upregulated during Edwardsiella tarda and Streptococcus agalactiae infection. Purified recombinant TroLEAP-2 (rTroLEAP-2) could significantly inhibit the in vitro growth of E. tarda and S. agalactiae. Overexpression of TroLEAP-2 in vivo was shown to significantly reduce E. tarda and S. agalactiae colonization of tissues, whereas its knockdown resulted in an increase of bacteria in fish tissues. We also saw that TroLEAP-2 overexpression significantly improved macrophage activation in vivo. Moreover, TroLEAP-2 can induce the expression of nonspecific immune-related genes. These results showed that it might play a significant role in the innate immune system of golden pompano. In conclusion, our results indicate that TroLEAP-2 plays an important role in antibacterial immunity and provides a new avenue for protection against pathogenic infections in golden pompano.

Small nucleolar RNA Sf-15 regulates proliferation and apoptosis of Spodoptera frugiperda Sf9 cells.

BACKGROUND: Small nucleolar RNAs (snoRNAs) function in guiding 2'-O-methylation and pseudouridylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). In recent years, more and more snoRNAs have been found to play novel roles in mRNA regulation, such as pre-mRNA splicing or RNA editing. In our previous study, we found a silkworm C/D box snoRNA Bm-15 can interact with Notch receptor gene in vitro. To further study the function of Bm-15, we cloned its homolog Sf-15 from Spodoptera frugiperda and investigate the function of Sf-15 in Sf9 cells. RESULTS: We showed that knocking down of Sf-15 can inhibit the proliferation, then induce apoptosis of insect S. frugiperda Sf9 cells, but the results were reversed when Sf-15 was overexpressed. De novo sequencing of transcriptome of Sf9 cells showed that the expression of 21 apoptosis-related genes were increased upon Sf-15 repression. Further analysis showed that a Ca2+-induced cell death pathway gene Cn (PPP3C, the serine/threonine-protein phosphatase 2B catalytic subunit), was significantly increased upon Sf-15 depression but decreased when Sf-15 was overexpressed, which indicated that Cn might be a potential target of Sf-15. CONCLUSIONS: We conclude that C/D box snoRNA Sf-15 can participate in apoptosis through regulating the expression of Ca2+-induced cell death pathway gene Cn in Sf9 cells. This is the first time that we found snoRNAs exhibiting dual functions in insect, which reveals a novel layer of ncRNA modulation in cell growth and death.

Experimental infection of BALB/c mice with a caprine Pestivirus H isolate.

To data, small animal Pestivirus H infection models have not been established. In order to develop a new infection model, BALB/c mice were inoculated with Pestivirus H strain HN1507. The virus-inoculated mice displayed nasal discharge and fever clinical signs. Histopathological changes in Pestivirus H-infected mice included alveolar septa thickening and alveolar atrophy in the lungs from 1 to 11 days post-inoculation (PI). Furthermore, we observed tracheal epithelial cell abscission and inflammatory cell infiltration in the tracheas from 1 to 9 days PI, infiltration of eosinophils in the spleens from 1 to 9 days PI, intestinal villi abscission and lysis of epithelial cells in the intestines from 1 to 11 days PI. The results of virus isolation showed that Pestivirus H replicated well in the lungs, tracheas, spleens, and intestines of infected BALB/c mice, and peak viral titers were observed 3 days PI. RT-PCR and immunofluorescence results were in agreement with the virus isolation results; however, the hearts of infected mice from 1 to 3 days PI were positive while virus isolation results were negative. To the best of our knowledge, this is the first study reporting Pestivirus H detection in BALB/c mice. Our findings indicated that Pestivirus H strain HN1507 was pathogenic to BALB/c mice and caused clinical signs and histopathological lesions in Pestivirus H-infected BALB/c mice.

Characterisation of Newly Emerged Isolates of Classical Swine Fever Virus in China, 2014-2015.

INTRODUCTION: In 2014-2015, the epidemic of classical swine fever (CSF) occurred in many large-scale pig farms in different provinces of China, and a subgenotype 2.1d of CSF virus (CSFV) was newly identified. MATERIAL AND METHODS: The phylogenetic relationship, genetic diversity, and epidemic status of the 2014-2015 CSFV isolates, 18 new CSFV isolates collected in 2015, and 43 other strains isolated in 2014-2015 were fully analysed, together with 163 CSFV reference isolates. RESULTS: Fifty-two 2014-2015 isolates belonged to subgenotype 2.1d and nine other isolates belonged to subgenotype 2.1b. The two subgenotype isolates showed unique molecular characteristics. Furthermore, the 2.1d isolates were found to possibly diverge from 2.1b isolates. CONCLUSION: This study suggests that the Chinese CSFVs will remain pandemic.

Ferritin from the Pacific abalone Haliotis discus hannai: Analysis of cDNA sequence, expression, and activity.

Ferritin plays an important role in iron homeostasis due to its ability to bind and sequester large amounts of iron. In this study, the gene encoding a ferritin (HdhFer2) was cloned from Pacific abalone (Haliotis discus hannai). The full-length cDNA of HdhFer2 contains a 5'-UTR of 121 bp, an ORF of 516 bp, and a 3'-UTR of 252 bp with a polyadenylation signal sequence of AATAAA and a poly(A) tail. It also contains a 31 bp iron-responsive element (IRE) in the 5'-UTR position, which is conserved in many ferritins. HdhFer2 consists of 171 amino acid residues with a predicted molecular weight (MW) ∼19.8 kDa and a theoretical isoelectric point (PI) of 4.84. The deduced amino acid sequence of HdhFer2 contains two ferritin iron-binding region signatures (IBRSs). HdhFer2 mRNA was detected in a wide range of tissues and was dominantly expressed in the gill. Infection with the bacterial pathogen Vibrio anguillarum significantly upregulated HdhFer2 expression in a time-dependent manner. Recombinant HdhFer2 (rHdhFer2) purified from Escherichia coli was able to bind ferrous iron in a concentration-dependent manner. In summary, these results suggest that HdhFer2 is a crucial protein in the iron-withholding defense system, and plays an important role in the innate immune response of abalone.

Molecular characterization of the cathepsin B of turbot (Scophthalmus maximus).

Cathepsin B is an enzymatic protein belonging to the peptidase C1 family. It is involved in diverse physiological and pathological functions that include immune response. In this study, we identified and characterized a cathepsin B homolog (SmCatB) from turbot (Scophthalmus maximus). SmCatB is composed of 330 amino acid residues and possesses typical domain architecture of cathepsin B, which contains a propeptide region and a cysteine protease domain, and the latter processes four conserved residues (Q101, C107, H277, and N297) in the active site. SmCatB shares 80.6-87.6% overall sequence identities with the cathepsin B of a number of teleost. SmCatB expression was detected in a wide range of tissues and upregulated by bacterial infection in a time-dependent manner. Recombinant SmCatB (rSmCatB-WT) purified from Escherichia coli exhibited apparent protease activity, which was optimal at 50 °C and pH 5.5. Compared to rSmCatB-WT, the mutant proteins rSmCatB-C107S, rSmCatB-H277A, and rSmCatB-N297A, which bear C107S, H277A, and N297A mutations, respectively, were significantly reduced in protease activity, with the highest reduction observed with rSmCatB-N297A. These results indicate that SmCatB is a bioactive protease that depends on the conserved structural features and that SmCatB is involved in pathogen-induced immune response.

HdhCTL1 is a novel C-type lectin of abalone Haliotis discus hannai that agglutinates Gram-negative bacterial pathogens.

C-type lectins (CTLs) are Ca(2+)-dependent carbohydrate recognition proteins, which play important roles in the innate immunity of both vertebrates and invertebrates. In this study, we identified and characterized a C-type lectin (named HdhCTL1) from Pacific abalone, Haliotis discus hannai. HdhCTL1 is composed of 176 amino acid residues and shares low (23.9%) identity with the known CTL of abalone. HdhCTL1 possesses a putative signal peptide and a carbohydrate-recognition domain (CRD) typical of CTLs. The CRD of HdhCTL1 contains four disulfide bond-forming cysteine residues that are highly conserved in CTLs. HdhCTL1 mRNA was detected in a wide range of tissues and expressed abundantly in the digestive gland. Experimental infection with the bacterial pathogen Vibrio anguillarum significantly upregulated HdhCTL1 expression in a time-dependent manner. Recombinant HdhCTL1 (rHdhCTL1) purified from Escherichia coli was able to agglutinate Gram-negative bacterial pathogens. The agglutinating ability of rHdhCTL1 was abolished in the presence of mannose. These results suggest that HdhCTL1 is a novel CTL which is likely to be involved in host defense against bacterial infection.

Macrophage migration inhibitory factor of Sciaenops ocellatus regulates immune cell trafficking and is involved in pathogen-induced immune response.

Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine involved in immunoregulation and inflammation. In this study, we examined the expression and biological function of a MIF, SoMIF, from red drum Sciaenops ocellatus. SoMIF is composed of 115 residues and shares 85-99% overall sequence identities with the MIF of a number of teleost. SoMIF expression was detected in a wide range of tissues and upregulated by bacterial and viral infection in a time-dependent manner. In head kidney (HK) leukocytes, pathogen infection induced SoMIF expression, and the expressed SoMIF was secreted into the extracellular milieu. Recombinant SoMIF (rSoMIF) purified from Escherichia coli inhibited the migration of both HK monocytes and lymphocytes, and this inhibitory effect was abolished by the presence of anti-rSoMIF antibodies. When rSoMIF was administered into red drum, it stimulated the production of reactive oxygen species in HK monocytes both in the presence and absence of pathogen infection. In vivo infection study showed that compared to untreated fish, fish pre-treated with rSoMIF before bacterial infection exhibited significantly lower bacterial loads in blood, kidney, spleen, and liver. Taken together, these results indicate that SoMIF is a secreted protein that regulates immune cell trafficking and is involved in pathogen-induced immune response.

Expression characterization and activity analysis of a cathepsin B from Pacific abalone Haliotis discus hannai.

Cathepsin B (EC 3.4.22.1) is a member of lysosomal cysteine protease and has a papain-like fold. In mammals, it is involved in protein degradation and other physiological processes including immune response. However, little is known about the function of cathepsin B in mollusks. In this study, we identified and analyzed a cathepsin B homolog (HdCatB) from Pacific abalone (Haliotis discus hannai), an economically important mollusk species cultured in East Asia. HdCatB is composed of 336 amino acid residues and its mature form is predicted to start at residue 86. HdCatB possesses typical domain architecture of cathepsin B and contains a propeptide region and a cysteine protease domain, the latter containing the four active site residues (Q108, C114, H282, and N302) that are conserved in many different organisms. HdCatB shares 40-60% overall sequence identities with the cathepsin Bofa number of vertebrates and invertebrates and is phylogenetically very close to mollusk cathepsin B. Quantitative real time RT-PCR analysis revealed that HdCatB expression occurred in multiple tissues and was upregulated by bacterial infection. Recombinant HdCatB purified from Escherichia coli exhibited apparent protease activity, which was optimal at 45 °C and pH 6.0. These results indicate that HdCatB is a bioactive protease that is likely to be implicated in the immune response of abalone during bacterial infection.

Identification and characterization of a cell surface scavenger receptor cysteine-rich protein of Sciaenops ocellatus: bacterial interaction and its dependence on the conserved structural features of the SRCR domain.

The scavenger receptor cysteine-rich (SRCR) proteins are secreted or membrane-bound receptors with one or multiple SRCR domains. Members of the SRCR superfamily are known to have diverse functions that include pathogen recognition and immunoregulation. In teleost, although protein sequences with SRCR structure have been identified in some species, very little functional investigation has been carried out. In this study, we identified and characterized a teleost SRCR protein from red drum Sciaenops ocellatus. The protein was named S. ocellatus SRCR1 (SoSRCRP1). SoSRCRP1 is 410-residue in length and was predicted to be a transmembrane protein, with the extracellular region containing a collagen triple helix repeat and a SRCR domain. The SRCR domain has six conserved cysteines, of which, C338 and C399, C351 and C409, and C379 and C389 were predicted to form three disulfide bonds. SoSRCRP1 expression was detected mainly in immune-relevant tissues and upregulated by bacterial and viral infection. In head kidney leukocytes, bacterial infection stimulated the expression of SoSRCRP1, and the expressed SoSRCRP1 was localized on cell surface. Recombinant SoSRCRP1 (rSoSRCRP1) corresponding to the SRCR domain was purified from Escherichia coli and found to be able to bind Gram-negative and Gram-positive bacteria. To examine the structure-function relationship of SoSRCRP1, the mutant proteins SoSRCRP1M1, SoSRCRP1M2, SoSRCRP1M3, and SoSRCRP1M4 were created, which bear C351S and C409S, C338S, C379S, and R325A mutations respectively. Compared to rSoSRCRP1, all mutants were significantly reduced in the ability of bacterial interaction, with the highest reduction observed with SoSRCRP1M4. Taken together, these results indicate that SoSRCRP1 is a cell surface-localized SRCR protein that binds bacterial ligands in a manner that depends on the conserved structural features of the SRCR domain.

A divalent DNA vaccine based on Sia10 and OmpU induces cross protection against Streptococcus iniae and Vibrio anguillarum in Japanese flounder.

Streptococcosis and vibriosis caused by Streptococcus iniae and Vibrio anguillarum respectively have affected fish culture industries around the world. Previous studies have indicated that the S. iniae antigen Sia10 and the V. anguillarum outer membrane protein OmpU, when used as DNA vaccines, induce protection in turbot (Scophthalmus maximus) and Asian seabass (Lates calcarifer) respectively. In this study, with an effort to develop effective vaccines against S. iniae and V. anguillarum, we constructed three DNA vaccines based on Sia10 and OmpU and examined their immune effects in a model of Japanese flounder (Paralichthys olivaceus), which in China is known to suffer from both streptococcosis and vibriosis. Of the three DNA vaccines constructed in this study, pIDSia10 and pIDOmpU express Sia10 and OmpU respectively, while pSiVa1 expresses Sia10 and OmpU as two individual proteins translated from a single bicistronic mRNA transcript. At 7 and 28 days post-vaccination, vaccine plasmids and expression of the vaccine-encoding genes were detected in the muscle, spleen, kidney, and liver of the vaccinated fish. Immunocolloidal gold electron microscopy detected production of Sia10 and OmpU proteins in pIDSia10- and pIDOmpU-vaccinated fish respectively, while both Sia10 and OmpU proteins were detected in pSiVa1-vaccinated fish. At one and two months post-vaccination, fish vaccinated with pIDSia10 and pSiVa1 exhibited comparable relative percent of survival (RPS) rates (80%-87%) following lethal S. iniae challenge. Similar protection rates were produced by fish vaccinated with pIDOmpU and pSiVa1 following lethal V. anguillarum challenge. Immunological analysis showed that (i) all vaccines induced specific serum antibody production which enhanced complement-mediated bactericidal activity, and (ii) pSiVa1 modulated the expression of a wide spectrum of immune relevant genes in a time-dependent manner. Together these results indicate that pSiVa1 is an effective bivalent vaccine that induces strong cross protection in flounder against S. iniae and V. anguillarum.