Characterization of a novel brain cell line from Jian carp (Cyprinus carpio var. Jian).

Jian carp (Cyprinus carpio var. Jian) is an economically important cultured fish in China. Currently, it is facing threats from infectious diseases including koi herpesvirus (KHV). Here, we established a new cell line, designated CCB-J, derived from the brain tissue of the Jian carp. CCB-J cells grew well in Leibovitz's L-15 medium containing 20% fetal bovine serum at 25 °C and have been subcultured for more than 60 passages. At the 30th passage, analysis showed that the number of chromosomes was 100, which is identical to that of other carp variants. Sequencing of the 18S ribosomal DNA confirmed that CCB-J originated from Jian carp. After transfection with the pEGFP-N1 plasmid, green fluorescence was observed in CCB-J. The replication of KHV in CCB-J cells was confirmed by RT-PCR and transmission electron microscopy. The viral titers of KHV in CCB-J cells and CCB cells, which have been widely used in the study of KHV, reached 103.9 and 101.8 median tissue culture infectious dose (TCID50/mL), respectively, within 14 days. The result of TaqMan PCR revealed that CCB-J cells were more sensitive to KHV than CCB cells. Meanwhile, a cytopathic effect (CPE) was also observed in the CCB-J cells in a shorter time post-infection compared with CCB cells. In summary, the CCB-J cell line will be a useful tool in the study of viral pathogenesis and vaccine research.

Characterization of novel antigenic vaccine candidates for nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae infection.

Streptococcus agalactiae is one of the important pathogens responsible for high mortality and economic losses of the tilapia industry worldwide. Based on ten serovars of S. agalactiae infection, subunit vaccine with conserved antigens is promising strategy corresponding stimulated long-term immunity and provides protection for animals against different serotypes of S. agalactiae. In the present study, eight proteins (AP, AL, LivK, ESAT6, essA, essB, essC and esaA) were selected from the S. agalactiae serotype Ia genome as immunogenic antigens with bioinformation and immune experiment assays. These recombinant proteins were successfully obtained through expression in Escherichia coli and the immunogenicity was assessed in tilapia challenge model. The results showed that the recombinant proteins caused high-level-specific antibodies production and high lysozyme activities, suggesting that the recombinant proteins induced specific humoral immune response and innate immune response of tilapia. The signficant increase were observed in the cytokines levels of TNF-α, IL-1ß, IFN-γ, cc1, cc2 and immune-related genes levels of CD8α and MHC factors in the spleen and head kidney tissues, suggesting that the recombinant proteins induced immune response of tilapia through cytokines signal pathway and activated high cytotoxic T-lymphocyte (CTL) activity of tilapia. Furthermore, vaccinated tilapia conferred high levels of protection against challenge with a lethal dose of highly virulent serovar Ⅰa (highest RPS was 91.60% in AL and essC protein groups). Our results indicated that the eight recombinant proteins induced high level of immune responses and offered protection against S. agalactiae infection, could be potential subunit vaccine candidates.

Oral vaccination using Artemia coated with recombinant Saccharomyces cerevisiae expressing cyprinid herpesvirus-3 envelope antigen induces protective immunity in common carp (Cyprinus carpio var. Jian) larvae.

Cyprinid herpesvirus 3 (CyHV-3) is the etiological agent of koi herpersvirus disease (KHVD), which causes serious economic losses in global common carp and ornamental koi carp production of larvae as well as adult type fish. To control KHVD, vaccines against CyHV-3 utilizing different immunization routes have been developed, among them, oral vaccination is the most desirable method to prevent fish diseases occurring at the early larval stage. Here, we developed an oral subunit vaccine through the Saccharomyces cerevisiae cell surface display of CyHV-3 envelope protein pORF65, then, the recombinant yeast fed to Artemia which served as bio-encapsulation vector by subsequently feeding the common carp (Cyprinus carpio var. Jian) larvae. The fluorescent observation showed that the Artemia and S. cerevisiae could deliver intact antigen to the hindgut of carp larvae suggesting the possibility of the vector for oral immunization. On this basis, after three immunizations at a week interval, the oral vaccine induced high level of specific anti-pORF65 antibody. Meanwhile, a significant difference of immune-related genes expression occurred including cxca, IL-1ß, IFN-a1, lysozyme, IgM and CD8α between vaccined group and blank control group. In addition, 30% of relative percent survival of carp larvae after immunization was obtained post the animal infection assay, offered an certain immune protection. Our results indicated that the oral pORF65 subunit vaccine bioencapsulated in Artemia induced the activation of immune response and high level of antibodies, which could be served as an oral vaccine candidate for the prevention of CyHV-3 infection.

Protective immunity against CyHV-3 infection via different prime-boost vaccination regimens using CyHV-3 ORF131-based DNA/protein subunit vaccines in carp Cyprinus carpio var. Jian.

Cyprinid Herpesvirus 3 (CyHV-3), also known as Koi Herpesvirus (KHV), causes Koi Herpesvirus Disease (KHVD) which leads to serious economic losses worldwide. To exploit DNA/subunit vaccine candidates, CyHV-3 ORF131 gene and cDNA was cloned and analyzed in the present study. Major B cell epitopes of deduced CyHV-3 pORF131 was also predicted. Then the complete CDS of CyHV-3 ORF131 was inserted into pEGFP-N1 vector and a modified pYD1/EBY100 system to construct the DNA and subunit vaccine, respectively. Subsequently, carp were immunized with homologous and heterologous prime-boost regimens relying on the constructed DNA and oral subunit vaccines. Then the protective immunity generated from different vaccines and regimens as well as the capacity of yeast (Saccharomyces cerevisiae) as an oral vaccine vehicle was evaluated. Our study confirmed that CyHV-3 ORF131 gene consisted of 2 introns and 3 exons encoding a 428 amino acids peptide. Further analysis indicated that four fragments of CyHV-3 pORF131 contained the major B cell epitopes (Cys20~Val140, Ser169~Tyr245, Thr258~Pro390, Phe414~Gln428), which could be linked and expressed in E. coli (BL21) as a truncated pORF131. The expression of full-length CyHV-3 pORF131 by pEGFP-N1 and yeast surface display was verified by In vitro assays before vaccination. Immunization of carp with CyHV-3 ORF131 DNA and subunit vaccines could evoke the activation of immune-related genes such as CXCa, CXCR1, IL-1ß, TNF-α, INF-a1, Mx-1, IgM, IgT1 and production of specific serum IgM measured by ELISA. RPS (relative percent of survival) ranging from 53.33% to 66.67% was acquired post challenge test. Moreover, flow cytometry analysis illustrated the delivery of surface-displayed CyHV-3 pORF131 to midgut after oral gavage. Thus, our findings suggest that CyHV-3 ORF131 can serve as DNA/subunit vaccines candidate and the yeast as an ideal oral vaccine vehicle.

LuxS/AI-2 in Streptococcus agalactiae reveals a key role in acid tolerance and virulence.

LuxS-mediated autoinducer-2 (AI-2) directly or indirectly regulates important physiologic function in a variety of bacteria. We found a luxS homologue in the genome of Streptococcus agalactiae, an important pathogen of tilapia. To investigate the relationship between luxS/AI-2 and pathogenicity for tilapia, its bioluminescent activity, acid resistance, cell adherence, virulence, and regulation of virulence gene were evaluated. Compared with the wild-type strain, the bioluminescent activity lost in the luxS mutant, its resistance to acid (pH2.8) was significantly decreased 33.8 times, and furthermore, its adherence to the NGF-2 cell line was dramatically reduced 3 times in the mutant strain. The virulence of the mutant strain was decreased in the tilapia infection model, exogenous AI-2 molecule (7.4nM) and luxS gene complementation with plasmid could complement the deficiencies of function in the luxS mutant strain. These results showed that inactivation of luxS gene caused a significant decrease of bioluminance, acid resistance, cell adhesion, virulence to tilapia and transcription levels of many virulence genes in S. agalactiae. Expression of the known stress resistance factors DnaK and GroEL, relative regulator factors CovR/CovS and virulence factor cpsE verified above results. These findings suggest that luxS may be involved in the interruption of bacterial virulence and resistance to environmental factors.

Protective efficacy of cationic-PLGA microspheres loaded with DNA vaccine encoding the sip gene of Streptococcus agalactiae in tilapia.

Streptococcus agalactiae (S. agalactiae) is an important fish pathogen, which has received more attention in the past decade due to the increasing economic losses in the tilapia industry worldwide. As existing effective vaccines of S. agalactiae in fish have obvious disadvantage, to select immunoprotective antigens and package materials would undoubtedly contribute to the development of novel oral vaccines. In the present study, surface immunogenic protein (sip) was selected from the S. agalactiae serovar I a genomes as immunogenic protein in DNA vaccine form with cationic chitosan and biodegradable and biocompatible PLGA. The pcSip plasmid in cationic-PLGA was successfully expressed in tissues of immunized tilapia and the immunogenicity was assessed in tilapia challenge model. A significant increase was observed in the cytokine levels of IL-1ß, TNF-α, CC1, CC2 in spleen and kidney tissues. Furthermore, immunized tilapia conferred different levels of protection against challenge with a lethal dose of highly virulent serovar I a S. agalactiae. Our results indicated that the pcSip plasmid in cationic-PLGA induced high level of antibodies and protection against S. agalactiae infection, could be effective oral DNA vaccine candidates.

Multiple Evolutionary Selections Involved in Synonymous Codon Usages in the Streptococcus agalactiae Genome.

Streptococcus agalactiae is an important human and animal pathogen. To better understand the genetic features and evolution of S. agalactiae, multiple factors influencing synonymous codon usage patterns in S. agalactiae were analyzed in this study. A- and U-ending rich codons were used in S. agalactiae function genes through the overall codon usage analysis, indicating that Adenine (A)/Thymine (T) compositional constraints might contribute an important role to the synonymous codon usage pattern. The GC3% against the effective number of codon (ENC) value suggested that translational selection was the important factor for codon bias in the microorganism. Principal component analysis (PCA) showed that (i) mutational pressure was the most important factor in shaping codon usage of all open reading frames (ORFs) in the S. agalactiae genome; (ii) strand specific mutational bias was not capable of influencing the codon usage bias in the leading and lagging strands; and (iii) gene length was not the important factor in synonymous codon usage pattern in this organism. Additionally, the high correlation between tRNA adaptation index (tAI) value and codon adaptation index (CAI), frequency of optimal codons (Fop) value, reinforced the role of natural selection for efficient translation in S. agalactiae. Comparison of synonymous codon usage pattern between S. agalactiae and susceptible hosts (human and tilapia) showed that synonymous codon usage of S. agalactiae was independent of the synonymous codon usage of susceptible hosts. The study of codon usage in S. agalactiae may provide evidence about the molecular evolution of the bacterium and a greater understanding of evolutionary relationships between S. agalactiae and its hosts.

Codon usage bias of the phosphoprotein gene of spring viraemia of carp virus and high codon adaptation to the host.

In this study, we calculated the relative synonymous codon usage (RSCU) value and the effective number of codons (ENC) value to carry out principal component analysis (PCA) and correlation analysis of the codon usage pattern of the phosphoprotein gene (P gene) of spring viraemia of carp virus (SVCV). The synonymous codon usage pattern in P genes is geography-specific, based on PCA analysis. The high correlation between (G + C)1,2 % and (G + C)3 % suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage and base components in P genes. At least 40 out of 59 synonymous codons are similarly selected in all functional genes within five complete SVCV genomes, and the hosts based on the RSCU data. These results not only provide insight into variations in the codon usage pattern of SVCV but also may help in understanding the processes governing the evolution of SVCV.