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.

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.

Improvement of specific growth rate of Pichia pastoris for effective porcine interferon-α production with an on-line model-based glycerol feeding strategy.

Effective expression of porcine interferon-α (pIFN-α) with recombinant Pichia pastoris was conducted in a bench-scale fermentor. The influence of the glycerol feeding strategy on the specific growth rate and protein production was investigated. The traditional DO-stat feeding strategy led to very low cell growth rate resulting in low dry cell weight (DCW) of about 90 g/L during the subsequent induction phase. The previously reported Artificial Neural Network Pattern Recognition (ANNPR) model-based glycerol feeding strategy improved the cell density to 120 g DCW/L, while the specific growth rate decreased from 0.15 to 0.18 to 0.03-0.08 h(-1) during the last 10 h of the glycerol feeding stage leading to a variation of the porcine interferon-α production, as the glycerol feeding scheme had a significant effect on the induction phase. This problem was resolved by an improved ANNPR model-based feeding strategy to maintain the specific growth rate above 0.11 h(-1). With this feeding strategy, the pIFN-α concentration reached a level of 1.43 g/L, more than 1.5-fold higher than that obtained with the previously adopted feeding strategy. Our results showed that increasing the specific growth rate favored the target protein production and the glycerol feeding methods directly influenced the induction stage. Consequently, higher cell density and specific growth rate as well as effective porcine interferon-α production have been achieved by our novel glycerol feeding strategy.

Sequence-related amplified polymorphism, an effective molecular approach for studying genetic variation in Fasciola spp. of human and animal health significance.

In the present study, a recently described molecular approach, namely sequence-related amplified polymorphism (SRAP), which preferentially amplifies ORFs, was evaluated for the studies of genetic variation among Fasciola hepatica, Fasciola gigantica and the "intermediate" Fasciola from different host species and geographical locations in mainland China. Five SRAP primer combinations were used to amplify 120 Fasciola samples after ten SRAP primer combinations were evaluated. The number of fragments amplified from Fasciola samples using each primer combination ranged from 12 to 20, with an average of 15 polymorphic bands per primer pair. Fifty-nine main polymorphic bands were observed, ranging in size from 100 to 2000 bp, and SRAP bands specific to F. hepatica or F. gigantica were observed. SRAP fragments common to F. hepatica and the "intermediate" Fasciola, or common to F. gigantica and the "intermediate" Fasciola were identified, excised and confirmed by PCR amplification of genomic DNA using primers designed based on sequences of these SRAP fragments. Based on SRAP profiles, unweighted pair-group method with arithmetic averages clustering algorithm categorized all of the examined representative Fasciola samples into three groups, representing the F. hepatica, the "intermediate" Fasciola, or the F. gigantica. These results demonstrated the usefulness of the SRAP technique for revealing genetic variability between F. hepatica, F. gigantica and the "intermediate" Fasciola, and also provided genomic evidence for the existence of the "intermediate" Fasciola between F. hepatica and F. gigantica. This technique provides an alternative and a useful tool for the genetic characterization and studies of genetic variability in parasites.

Improving Performance and Operational Stability of Porcine Interferon-α Production by Pichia pastoris with Combinational Induction Strategy of Low Temperature and Methanol/Sorbitol Co-feeding.

Various induction strategies were investigated for effective porcine interferon-α (pIFN-α) production by Pichia pastoris in a 10 L fermenter. We found that pIFN-α concentration could be significantly improved with the strategies of low-temperature induction or methanol/sorbitol co-feeding. On this basis, a combinational strategy of induction at lower temperature (20 °C) with methanol/sorbitol co-feeding has been proposed for improvement of pIFN-α production. The results reveal that maximal pIFN-α concentration and antiviral activity reach the highest level of 2.7 g/L and 1.8 × 10(7) IU/mg with the proposed induction strategy, about 1.3-2.1 folds higher than those obtained with other sub-optimal induction strategies. Metabolic analysis and online multi-variable measurement results indicate that energy metabolic enrichment is responsible for the performance enhancement of pIFN-α production, as a large amount of ATP could be simultaneously produced from both formaldehyde oxidation pathway in methanol metabolism and tricarboxylic acid (TCA) cycle in sorbitol metabolism. In addition, the proposed combinational induction strategy enables P. pastoris to be resistant to high methanol concentration (42 g/L), which conceivably occur associating with the error-prone methanol over-feeding. As a result, the proposed combinational induction strategy simultaneously increased the targeted protein concentration and operational stability leading to significant improvement of pIFN-α production.

Enhancing pIFN-α production and process stability in fed-batch culture of Pichia pastoris by controlling the methanol concentration and monitoring the responses of OUR/DO levels.

Effective expression of porcine interferon-α (pIFN-α) with recombinant Pichia pastoris was conducted in a bench-scale fermentor using an in situ methanol electrode-based feeding process with the control level of methanol concentration linearly increased to 10 g l⁻¹ for the first 20 h and maintained at 10 g l⁻¹ for the rest of expression phase. With this two-stage control process, the highest pIFN-α concentration reached a level of 1.81 g l⁻¹, which was 1.5-fold of that in the previous constant 10 g l⁻¹ induction experiments. There is an improvement of the pIFN-α productivity from more distribution of carbon flux to protein expression. The pIFN-α expression stability could be further enhanced by a simple on-line fault diagnosis method for methanol overfeeding based on oxygen uptake rate changing patterns. By implementing corrective action of feeding glycerol after fault detection, the production yield increased to twice the amount it would have been without the diagnosis.

[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.