Immortalization of chicken embryonic liver-derived cell line by stable expression of hMRP18S-2 for serotype 4 fowl adenovirus propagation.

Inclusion body hepatitis and hydropericardium-hepatitis syndrome caused by serotype 4 fowl adenovirus (FAdV-4) have emerged in China since 2013. FAdV is usually propagated in primary chicken embryonic liver cells or embryo yolk sac. The aim of this work was to develop an immortalized CEL cell line by stable expression of human mitochondrial ribosomal protein 18S-2, named CEL-hMRP18S-2 cells, for the propagation of FAdV-4. The maximum cell density of CEL-hMRP18S-2 cells could reach 2.65 × 106 cells/ml in four-days culture. According to the mRNA levels of cell-cycle related genes in CEL-hMRP18S-2 cells tested by qRT-PCR, we speculated that the transformation of hMRP18S-2 into CEL cells caused the functional inactivation of p53 and the significant down-regulation of p15INK4b might cause the hyperphosphorylated form of Rb, releasing E2F-1 factor and enhancing the E2F-dependent transcription for cell cycle progression. It was suspected that the up-regulated c-Myc mRNA level at the initial period of immortalization might prompt transformed cells through the G0-G1 checkpoint. The normal CPE was observed in CEL-hMRP18S-2 cells infected by FAdV-4 and microcarrier suspension culture performed for FAdV-4 propagation with 9.0 lgTCID50/ml suggested that CEL-hMRP18S-2 cells could be a useful continuous cell line for isolation of wild FAdV and production of FAdV-inactivated vaccine.

☆DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus.

Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics.

Enhanced soluble production of cholera toxin B subunit in Escherichia coli by co-expression of SKP chaperones.

The cholera toxin B subunit (CTB) is a nontoxic portion of the cholera toxin that retains mucosal adjuvant properties. Expression of CTB in Escherichia coli is difficult as CTB aggregates and accumulates as insoluble inclusion bodies. To remedy this problem, the periplasmic chaperone, SKP, was investigated as possible co-expression partner to increase the solubility of recombinant CTB (rCTB) in E. coli. The result showed co-expression of SKP enhanced the soluble expression of rCTB in E. coli. Moreover, soluble rCTB was successfully expressed and secreted into the periplasmic space through the direction of the LTB leader signal. rCTB in periplasm was purified using an immobilized d-galactose resin; GM1-ELISA experiments showed that rCTB retains strong GM1 ganglioside-binding activity. Intranasal administration of ovalbumin (OVA) with rCTB significantly induced both mucosal and humoral immune responses specific to OVA. These data indicate that co-expression of the molecular chaperone SKP with CTB increased the solubility of rCTB while maintaining its function.

[A novel double expression shuttle vector to get marker-free recombinant modified vaccinia virus Ankara].

UNLABELLED: A novel double expression shuttle vector named pLR-gpt was constructed for marker-free recombinant modified vaccinia virus Ankara generation. A delectable Eco gpt marker was adopted with Cre/LoxP DNA recombination system and a BHK-21 cell line that can express Cre enzyme. Eco gpt gene controlled by P7.5 promoter from Vaccinia virus was cloned between two LoxP sites in the same direction. Additionally, two multiple cloning site under control of other two Vaccinia virus promoters were constructed outside LoxP sites. With this new transfer vector, Eco gpt marker in rMVA can be deleted on BHK-Cre with interaction between Cre enzyme and LoxP sequence. In order to verify the efficacy of this system, ORF5 and ORF6 gene of Porcine reproductive and respiratory syndrome virus (PRRSV) NJ-a strain were cloned into two multiple cloning sites of pLR-gpt to construct recombinant plasmid pLR-ORFS/ORF6. Homologous recombination between pLR-ORF5/ORF6 and wtMVA on BHK-21 cell was mediated by liposome by infecting cells with 0.01 MOI wtMVA two hours before transfection. After twelve cycles of selection, recombinant MVA with selecting marker Eco gpt was obtained and named as rMVAgpt-GP5/M. By infecting BHK-Cre, the Eco gpt marker in rMVAgpt-GP5/M was deleted and this rMVA was named as rMVA-GP5/M. Expression of GP5 and M protein was identified with Western blotting and IFA. Results from PCR and biological study for rMVA indicated that Eco gpt marker was completely deleted. CONCLUSIONS: double expression transfer vector for marker-free recombinant Modified vaccinia virus Ankara generation was successfully constructed, and works well in MVA expression system.

Co-expressing GP5 and M proteins under different promoters in recombinant modified vaccinia virus ankara (rMVA)-based vaccine vector enhanced the humoral and cellular immune responses of porcine reproductive and respiratory syndrome virus (PRRSV).

The porcine reproductive and respiratory syndrome virus (PRRSV) has three major structural proteins which designated as GP5, M, and N. Protein GP5 and M have been considered very important to arouse the humoral and cellular immune responses against PRRSV infection and proposed to be the excellent candidate proteins in the design of PRRS bioengineering vaccine. There were some attempts on expressing GP5 or M in DNA vaccine and adenovirus to arouse humoral and cellular immune responses, but few papers have been reported on that the immune response can be difference because of the expression patterns of GP5 and M proteins in the recombinant virus. In this article, four recombinant viruses that expressed GP5 and M proteins of PRRSV in the modified vaccinia virus ankara (MVA) with different expression patterns were made. In these recombinant virus (rMVAs), GP5 and M proteins were expressed in MVA in the same virus but under the control of two promoters (rMVA-GP5/M), or as a fusion protein under one promoter (rMVA-GP5-M), or separately (rMVA-GP5 and rMVA-M). The humoral and cellular immune responses for the four recombinant viruses were evaluated with mouse model. Every mouse was inoculated with 5 x 10(5) TCID50 of the different rMVAs and boosted 3 weeks later. Neutralizing antibody titers for each group were detected with virus neutralization test assay weekly after the primary inoculation for 13 weeks to evaluate the humoral immune response. The production of gamma interferon (IFN-gamma), interleukin-2 (IL-2), and interleukin-4 (IL-4) was detected in splenocytes of rMVA-inoculated mice at 30, 60, and 90 days post inoculation to evaluate the cellular immune response. Results showed that rMVA-GP5 and rMVA-M cannot induce obvious humoral and cellular immune responses; rMVA-GP5-M inoculated group developed better immune responses than rMVA-GP5 and rMVA-M inoculated groups; however, mice inoculated with rMVA-GP5/M maintained the strongest cellular response against PRRS and consistently enhanced the anti-PRRSV humoral responses. The strategy of co-expressing PRRSV GP5 and M protein in MVA under the control of different promoters might be an attractive method for future PRRSV vaccine design.

[Construction and the immunogenicity of the recombinant Modified Vaccinia Virus Ankala co-expressing ORF4, ORF5 and ORF6 genes of porcine reproductive and Respiratory Syndrome Virus NJ-a strain].

To develop investigate the recombinant MVA(rMVA) vaccines against PRRSV infection, the ORF4, ORF5 and ORF6 of PRRSV NJ-a strain were subcloned into the MVA transfer vector p II LR and the resultant recombinant vector was called p II LR-ORF4/ORF5/ORF6. The rMVA was generated by transfecting MVA-infected BHK-21 cells with the recombinant vector and screened by plaque purification after X-gal staining. After six rounds of purification, insertion of PRRSV GP4, GP5 and M genes into the MVA genome was confirmed by PCR analysis and expression of the three proteins was identified by Western-blot and IFA. Each of the tested mice was inoculated with 5 x 10(5) TCID(50)/mouse of the rMVA-GP4/GP5/M and boosted 3 weeks later. Neutralization assay showed that PRRSV-specific neutralizing antibodies were detectable at 3 weeks and reached the highest titer (2(5)) by 8 weeks after the primary vaccination, which maintained stable until the end of the experiment. The significant lymphocyte proliferation responses were also observed in mice immunized with rMVA-GP4/GP5/M. These results indicate the rMVA co-expressing PRRSV ORF4, ORF5 andORF6 genes may be an attractive candidate vaccine for preventing PRRSV infection.

[Influence of epitope A modification and N-linked glycosylated site mutation of PRRSV NJ-a strain ORF5 gene on the ability to induce neutralizing antibodies and T cell proliferation response].

To enhance the DNA immunogencity of PRRSV ORF5 gene, CpG sequence and the universal helper T cell antigen epitope (PADRE) sequence were inserted between the decoy epitope and the neutralizing epitope. At the same time, site-mutations were introduced at N33 and N51 to diminish the coverage effect to epitope B from the polysaccharides. Subsequently, the modified ORF5 gene (MORF5) and PRRSV ORF6 gene were cloned into the eukaryotic expression vector pcDNA3.0 under the control of two CMV promoters, respectively. With indirect immunofluorescence assay and Western-blot the expression in vitro of the two genes was confirmed, then six-week-old Balb/C mouse were immunized with the modified expression plasmid pcDNA-M5A-6A. The non-modified expression plasmid pcDNA-5A-6A, the blank eukaryotic expression plasmid pcDNA3.0, living attenuated vaccine and inactivated vaccine were used as controls. The PRRSV specific neutralizing antibodies and the T cell proliferation response were elevated with virus neutralization assay and MTf method. Results indicate that the modified plasmid pcDNA-M5A-6A can elicit not only higher titer of neutralizing antibodies in a rapid time, but also more vigorous T cell proliferation response compared with the non-modified plasmid pcDNA-5A-6A and commercial vaccines, indicating that DNA vaccine pcDNA-M5A-6A maybe a promising candidate for PRRS prevention.

Potential use of a transposon Tn916-generated mutant of Aeromonas hydrophila J-1 defective in some exoproducts as a live attenuated vaccine.

Aeromonas hydrophila is a Gram-negative opportunistic pathogen that causes disease in a wide range of hosts due to its multifactorial virulence. Here we describe the application of transposon insertion mutagenesis approach to obtain an exoenzyme mutant of A. hydrophila strain J-1. Immunization of swordtail fish (Xiphophorus helleri Heckel) with the highly attenuated mutant provided protection (survival of 27 out of 35 fish, compared to survival of only 13 out of 35 control fish) in the fish given the highest immunization dose (10(7) CFU) against intraperitoneal challenge with the wild J-1 strain. Immunization with doses of 10(5) or 10(3) did not provide significant protection.