Development of an epitope-based competitive ELISA for the detection of antibodies against Tibetan peste des petits ruminants virus.

AIMS: To develop an effective diagnostic kit, based on a competitive ELISA-based system (cELISA), for detecting serum antibody against peste des petits ruminants virus (PPRV). METHODS: Epitope peptides of the nucleocapsid (N) protein of Tibetan PPRV were synthesized chemically and injected into rabbits to prepare hyperimmune antisera. Test sera were incubated simultaneously with hyperimmune antisera and added to the wells of ELISA plates coated previously with recombinant N protein. Horseradish peroxidase-conjugated goat anti-rabbit antibody was employed to detect the quantity of hyperimmune antisera combined with recombinant N protein. RESULTS: A cELISA has been developed for monitoring PPRV infections with a cutoff value of 35. Relative sensitivity and specificity values of the epitope-based cELISA were 96.18 and 91.29%, respectively, when compared with a commercial cELISA kit in a test involving 1,039 serum samples. CONCLUSION: We report an efficient method for preparing antibody suitable for incorporation into a cELISA that can be used routinely for the detection of PPRV antibodies in serum samples. The method eliminated the requirement for virus culture and monoclonal antibody preparation, reduced the biorisk posed by virus-dependent manipulations, and the performance of the resultant cELISA compared favorably with a commercially available cELISA kit.

Methanol/sorbitol co-feeding induction enhanced porcine interferon-α production by P. pastoris associated with energy metabolism shift.

The production of porcine interferon-α (pIFN-α) by Pichia pastoris was largely enhanced when adopting sorbitol/methanol co-feeding induction strategy at 30 °C in a 10-L fermentor. Analysis of energy regeneration pattern and carbon metabolism revealed that major energy metabolism energizing pIFN-α synthesis shifted from formaldehyde dissimilatory energy metabolism pathway to TCA cycle under the methanol/sorbitol co-feeding induction strategy. The sorbitol/methanol co-feeding induction strategy weakened formaldehyde dissimilatory pathway and repressed the accumulation of toxic metabolite-formaldehyde, reduced theoretical oxygen consumption rate and oxygen supply requirement, and increased energy/methanol utilization efficiency so that more methanol could be effectively used for pIFN-α synthesis. As a result, pIFN-α antiviral activity reached a highest level of 1.8 × 10(7) IU/mL which was about 10- to 200-folds of those obtained under pure methanol induction at 20 and 30 °C, respectively.

Development of an indirect ELISA with artificially synthesized N protein of PPR virus.

The full-length gene encoding the nucleocapsid (N) protein of the virus (PPRV) responsible for an outbreak of peste des petits ruminants in Tibet in 2007 was synthesized in two stages using overlapping PCR without the need for viral genomic cDNA as template. The full-length N gene was successfully expressed in Escherichia coli, and the purified gene product bound to monoclonal antibody raised against PPRV N protein. Furthermore, it was able to replace recombinant B-N antigen as the coating antigen in a commercial ELISA kit prepared with another PPRV strain. Recombinant protein was employed as the coating antigen to develop an indirect ELISA for PPRV antibody detection in the sera of infected small ruminants. Antibody detection was optimal at a 1:200 serum dilution and an antigen concentration of 3.2 µg/ml, and the positive threshold (cutoff) value of the assay was 2.18. Analysis of 697 serum samples revealed the sensitivity and specificity of the indirect ELISA to be 96.7 and 96.1%, respectively, compared with a commercially available ELISA test.

[Quasispecies investigation on swine hepatitis E viruses].

The objective of current study was to investigate the quasispecies of hepatitis E virus in swine. The partial ORF2 region of HEV envelope gene from four swine HEV strains was amplified by RT-nested polymerase chain reaction (RT-nPCR). After cloning and transformation of PCR products, 20 positive clones of each HEV isolate were subject to sequencing and DNA analysis. The homology among the different clones of each isolates was 96.8%-99.7%, 98.8%-99.7%, 98.8%-99.7% and 100%, respectively, while there was 96.8%-100% sequence identity at the nucleotide level compared with HEV strains isolated in Shanghai (SAAS-JDY5). This study confirmed that there existed quasispecies of HEV in swine.

Full genomic sequence analysis of swine genotype 3 hepatitis E virus isolated from Shanghai.

The full genomic nucleotide sequence of a previously identified genotype 3 hepatitis E virus (HEV), strain SAAS-JDY5, was obtained using RT-PCR and rapid amplification of cDNA ends (RACE). The genome consisted of 7225 nucleotides, excluding a poly-A tail at the 3' terminus, and contained three open reading frames (ORFs), ORF-1, ORF-2 and ORF-3, encoding 1702, 660 and 113 amino acids, respectively. Phylogenetic analysis confirmed that SAAS-JDY5 belonged to genotype 3 HEV and was most closely related to the Japanese isolate wbJYG1 (AB222184). SAAS-JDY5 shared approximately 87% nucleotide similarity to human and swine strains from the United States, compared with 74-75% similarity to Asian (genotype 4) and Mexican strains (genotype 2). Alignment of the SAAS-JDY5 genomic sequence with reference sequences of the same genotype revealed one nucleotide substitution and one deletion at positions 5145 and 7189 (3' UTR), respectively. Moreover, SAAS-JDY5 contained two additional nucleotides (AC) at the very end of the 3'-terminus preceding the poly-A tail of the genome. Comparison of the putative amino acid sequence encoded by the SAAS-JDY5 genome with sequences of other genotype 3 isolates revealed 15 unique amino acid substitutions and one deletion in ORF-1, and three substitutions in ORF-2.

[Generation of Chicken Germ-line Chimeras by Transferring PGCs and Their Identification by AFLP.].

PGCs (Primordial germ cells) were isolated from the blood of 51~56 h hatching Shiqiza chicken embryos by Ficoll density gradient centrifugation. The PGCs were injected into 2.5 d hatching embryos of H breed chicken to produce germ-line chimeras. AFLP checking method was established to identify chicken germline chimeras. Eight germ-line H-S chimera embryos were identified among 20 developing H breed embryos.

[High level secretion expression of PoIFNalpha in Pichia pastoris].

The porcine alpha interferon gene was inserted into the Pichia pastoris expression vector of pPICZalphaA which contains AOX I promoter and alpha-factor signal sequence. The recombinant plasmid was transformed into host cell E.coli JM109 and then was extracted for analysis of restriction enzymes. It was confirmed that heterogeneous gene spliced into vector pPICZalphaA was IFNalpha gene. The recombinant plasmid of pPICZalphaA-IFNalpha was linearnized by Sac I and transformed into KM71 by electroporation. SDS-PAGE and Western blot analysis showed that IFNalpha product was observed in the supernants with a little larger molecular weight size than the natural IFNalpha. The rIFN gene has the same antigenicity as natural one. The expressed rIFN accumulated up to about 0.45mg/mL. The cytokine activity of the supernantants was vertified by WISH/VSV system,which is about 2.1x10(4)IU/mL.

[Site-directed mutation of PoIFN-alpha and its expression in Escherichia coli].

By using huge primer PCR Cys86 (TGC) of PoIFN-alpha was mutated to Tyr(TAC), and the first code TGT was simultaneously changed to TGC, which is a bias code of E. coli. The expression plasmid pGEX-IFN was constructed successfully. Recombinant porcine IFN alpha, which is expressed as inclusion bodies, was about 20% of the total proteins. The inclusion body was dissolved in 8 mol/L urea and subsequently renatured by dilution in refolding buffer. In order to obtain pure protein, the renatured IFN alpha was purified by FPLC, and the cytokine activity (5200 IU/mg) was verified by inhibiting the cytopathic effect.