[Show abstract][Hide abstract] ABSTRACT: Various induction strategies were investigated for effective porcine interferon- (pIFN-) production by Pichia pastoris in a 10 L fermenter. We found that pIFN- 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- production. The results reveal that maximal pIFN- concentration and antiviral activity reach the highest level of 2.7 g/L and 1.8×107 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 on-line multi-variables 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 Pichia 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.
Applied Biochemistry and Biotechnology 03/2015; DOI:10.1007/s12010-015-1590-6 · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: New epidemic broke out in recent year which was suspected to be caused by variant Muscovy duck parvovirus (MDPV). For this reason, new MDPV detection methods are needed for the new virus strains. In this study, a pair of primers were designed according to the full-length genome of MDPV strain SAAS-SHNH, which were identified in 2012, and were used to amplify the vp3 gene of MDPV by polymerase chain reaction. After being sequenced, the vp3 gene was subcloned into the prokaryotic expression vector PET28a. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. SDS-PAGE and Western blotting analysis showed the MDPV vp3 gene was successfully expressed. After being purified by Ni2+ affinity chromatography system, the recombinant protein was used as antigen to immunize rabbits to obtain antiserum. Western blotting analysis showed that the acquired antiserum could react specifically with VP3 protein of J3D6 strain and MDPV vaccine strain. The antiserum could also be used for detection of cultured MDPV from primary duck embryo fibroblasts by immune fluorescence assay (IFA). It could be concluded that the VP3 protein and its antibody prepared in the research could be used for detection of VP3 antiserum and antigen respectively.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 01/2015; 31(1):65-74.
[Show abstract][Hide abstract] ABSTRACT: The full-length genome of strain SAAS-SHNH, a MDPV isolated from Muscovy Duck in Shanghai, has been sequenced and shown to share 93.7% nucleotide identity with MDPV strain FM (NC_006147). Two putative genetic recombination events were identified as occurring within the 419∼610 nt and 3113∼4241 nt regions of the SAAS-SHNH genome which, for the first time, provide evidence of recombination between MDPVs and GPVs.
[Show abstract][Hide abstract] ABSTRACT: Nucleoprotein (NP) is the most abundant and highly immunogenic protein of morbillivirus, and is presently the basis of most diagnostic assays for peste des petits ruminants virus (PPRV). In this study, fine epitope mapping and conservation analysis of linear B-cell epitopes on the PPRV NP has been undertaken using biosynthetic peptides. Nineteen linear B-cell epitopes were identified and their corresponding minimal motifs were located on the NP of PPRV China/Tibet/Geg/07-30. Conservation analysis indicated that ten of the 19 minimal motifs were conserved among 46 PPRV strains. Peptides containing the minimal motifs were recognized using anti-PPRV serum from a goat immunized with PPRV vaccine strain Nigeria 75/1. Identified epitopes and their motifs improve our understanding of the antigenic characteristics of PPRV NP and provide a basis for the development of epitope-based diagnostic assays.
[Show abstract][Hide abstract] ABSTRACT: A cDNA clone of a genotype 4 swine hepatitis E virus (HEV) strain (SAAS-FX17), identified in Shanghai, has been constructed. Capped RNA transcripts were prepared in vitro and shown to be replication-competent in Huh7 cells. Sprague-Dawley (SD) rats administered the RNA transcripts by intrahepatic inoculation developed active infections as evidenced by fecal virus shedding and sero-conversion to anti-HEV. The former was first detected between 23 and 30 days post-inoculation (dpi) and persisted until 45dpi. Sera of rats inoculated with RNA transcripts became anti-HEV positive between 30 and 40dpi, and reverted to anti-HEV negative at 52dpi. Our data indicate for the first time that intrahepatic inoculation of rats with RNA transcripts of an HEV cDNA clone may serve as an alternative animal model for HEV research.
[Show abstract][Hide abstract] ABSTRACT: Background
Four major genotypes of hepatitis E virus (HEV), the causative agent of hepatitis E, have so far been recognized. While genotypes 3 and 4 are both zoonotic, the disease symptoms caused by the latter tend to be more severe. To examine if specific nucleotide/amino acid variations between genotypes 3 and 4 play a role in determining the severity of hepatitis E disease, the complete genome of one swine HEV genotype 4 isolate, SAAS-FX17, was determined and compared with other genotype 4 and genotype 3 genomes to identify putative HEV genotype 4 virulence determinants.
A total of 42 conformable nt/aa variations between genotype 3 and 4 HEVs were detected, of which 19 were proposed to be potential disease severity determinants for genotype 4 strains.
One potential determinant was located in each of the 5'-UTR and 3'-UTR, 3 and 12 within ORF1 and ORF2 respectively, and 2 in the junction region.
[Show abstract][Hide abstract] ABSTRACT: Porcine interferon-alpha (pIFN-alpha) fermentative production by recombinant Pichia pastoris was carried out in a 10-L bioreactor to study its metabolism changes and effects on fermentation under different inducing strategies, by analyzing the change patterns of the corresponding metabolism and energy regeneration. The results show that the specific activities of alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH) largely increased when reducing temperature from 30 degrees C to 20 degrees C under pure methanol induction, leading significant enhancements in methanol metabolism, formaldehyde dissimilatory energy metabolism and pIFN-alpha antiviral activity. The highest pIFN-alpha antiviral activity reached 1.4 x 10(6) IU/mL, which was about 10-folds of that obtained under 30 degrees C induction. Using methanol/sorbitol co-feeding strategy at 30 degrees C, the major energy metabolism energizing pIFN-alpha synthesis shifted from formaldehyde dissimilatory energy metabolism pathway to TCA cycle, formaldehyde dissimilatory pathway was weakened and accumulation of toxic intermediate metabolite-formaldehyde was relieved, and methanol flux distribution towards to pIFN-alpha synthesis was enhanced. Under this condition, the highest pIFN-alpha antiviral activity reached 1.8 x 10(7) IU/mL which was about 100-folds of that obtained under pure methanol induction at 30 degrees C. More important, enhanced pIFN-alpha production with methanol/sorbitol co-feeding strategy could be implemented under mild conditions, which greatly reduced the fermentation costs and improved the entire fermentation performance.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 02/2012; 28(2):164-77.
[Show abstract][Hide abstract] ABSTRACT: The inhibitory effects of recombinant porcine interferon alpha (rPoIFN-α) on the propagation of low-virulence PRRSV (lvPRRSV) in MARC-145 cells, and on the progress and severity of high virulence PRRSV (hvPRRSV)-induced infections in pigs, were determined. Pre-treatment of MARC-145 cells with increasing concentrations of rPoIFN-α prior to infection with lvPRRSV decreased the observed cytopathic effects (CPEs) in a concentration-dependent manner. Viral propagation and antibody response were temporarily delayed in swine treated with rPoIFN-α either at the same time as the hvPRRSV challenge was administered or post-challenge. Exposure of challenged animals to rPoIFN-α after the onset of disease symptoms alleviated associated hyperthermia. Variations in lymphocyte subsets indicated that rPoIFN-α treatment might alleviate damage to the immune system or enhance propagation of host cytotoxic T-lymphocytes when the treatment was applied simultaneously with the virus or 1dpc, respectively.
Research in Veterinary Science 01/2012; 93(2):1060-5. DOI:10.1016/j.rvsc.2011.12.006 · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hepatitis E virus (HEV) causes acute or fulminant hepatitis in humans and is an important public health concern in many developing countries. China has a high incidence of HEV epidemics, with at least three genotypes (1, 3 and 4) and nine subtypes (1b, 1c, 3b, 4a, 4b, 4d, 4g, 4h and 4i) so far identified. Since genotype 3 and the newly identified subtype 4i have been exclusively limited geographically to Shanghai and its neighboring provinces, the epidemiology of HEV infections within the municipality, a major industrial and commercial center, deserves closer attention.
A total of 65 sequences, 60 located within the HEV SH-SW-zs1 genome [GenBank:EF570133], together with five full-length swine and human HEV genomic sequences, all emanating from Shanghai, were retrieved from GenBank. Consistent with the primary role of genotype 4 in China overall, analysis of the sequences revealed this to have been the dominant genotype (58/65) in Shanghai. Six HEV subtypes (3b, 4a, 4b, 4d, 4h and 4i) were also represented. However, although subtype 4a is the dominant subtype throughout China, subtype 4i (29/65) was the most prevalent subtype among the Shanghai sequences, followed by subtypes 4d (10/65) and 4h (9/65). Subtypes 4h, 4i and 4d were found in both swine and humans, whereas 4b was found only in swine and subtype 4a only in humans.
Six different swine and human HEV subtypes have so far been documented in Shanghai. More molecular epidemiological investigations of HEV in swine, and particularly among the human population, should be undertaken.
[Show abstract][Hide abstract] ABSTRACT: We studied the distribution and development of hepatitis E virus (HEV) genotypes 3 and 4 in pig farms located within the Shanghai metropolitan area. A total of 1,487 swine fecal samples were collected from 39 pig farms during 2009-2010. The average incidence rates for genotype 3 and genotype 4 HEV were 10.6 and 9.3%, respectively. The frequencies of genotype 3 and genotype 4 HEV among the farms were inversely related (r = -0.7423), suggesting that the two genotypes competed within the environmental niche provided by the porcine host. In addition, all of the farms tested positive for both genotype 3 and genotype 4 HEV, indicating that the former is becoming more prevalent in the Shanghai area. The overall HEV incidence rate during 2009-2010 was 20%, which supports the existence of homeostasis within the porcine HEV reservoir.
[Show abstract][Hide abstract] ABSTRACT: The performance of traditional heterologous protein production by Pichia pastoris with methanol induction at 30 °C is poor, characterized by low ATP regeneration rate and weak operation stability. A low
temperature induction strategy at 20 °C was thus adopted for efficient porcine interferon-α production in a 10 L fermentor. With the strategy, maximal methanol tolerance level could reach about 40 g/L to effectively
deal with methanol concentration variations, so that the complicated on-line methanol measurement system could be eliminated.
Moreover, metabolic analysis based on multiple state-variables measurements indicated that pIFN-α antiviral activity enhancement profited from the formation of an efficient ATP regeneration system at 20 °C induction. Compared
to the induction strategy at 30 °C, the proposed strategy increased the ATP regeneration rate by 49–66%, the maximal pIFN-α antiviral activity was enhanced about 20-fold and reached a higher level of 1.5×106 IU/mL.
Key wordsFed-batch Culture–Fermentation–
Korean Journal of Chemical Engineering 06/2011; 28(6):1412-1419. DOI:10.1007/s11814-010-0527-6 · 1.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Efficient porcine interferon-alpha (pIFN-alpha) expression in high density recombinant Pichia pastoris cultivation was achieved in a 5 l bench-scaled bioreactor. The results indicated that a high and stable oxygen uptake rate (OUR) during induction phase was closely related with pIFN-alpha production efficiency. The multi-variables clustering and analysis results showed that the achievement of a high and stable OUR relied on a higher glycerol consumption rate during fed-batch culture phase and a moderate methanol level (around 10 g/l) during induction phase. In the high and stable OUR environments (200-300 mmol/l/h), the highest pIFN-alpha antiviral activity could reach a level of 6.7 x 10(6) IU/ml, which was more than 10-300-folds higher than those obtained at lower OUR (80-200 mmol/l/h) using the same bioreactor and those obtained in shaking flasks. Clustering and analysis of the specific growth and glycerol consumption rates data during culture phase could detect the ill fermentation state at early stage, potentially provided a simple and effective fault alarming/diagnosis method for the achievement of stable pIFN-alpha production.
[Show abstract][Hide abstract] ABSTRACT: A total of 493 fecal samples collected from local Shanghai pig farms were examined for Hepatitis E virus (HEV) after the introduction of stricter sanitary measures following outbreaks of a high fever-associated pig disease during 2006 and 2007. Our investigation revealed that, while the overall occurrence of HEV RNA positives decreased by only 3.7%, the incidence of HEV genotype 4 increased from 9.8% to 20.6% whereas the incidence of HEV genotype 3 decreased from 16.2% to 1.6%. As well as demonstrating that HEV genotype 3 was more sensitive than genotype 4 to the stricter sanitation procedures, our data also suggested that a homeostasis mechanism, whereby the overall incidence of HEV is maintained at a specific population level, might exist in the porcine HEV reservoir. Furthermore, in one case, we encountered the coexistence of HEV genotypes 3 and 4 within the same sample, indicating the possibility of future HEV infections of increased severity and even the occurrence of a HEV pandemic due to genetic recombination and species evolution.
[Show abstract][Hide abstract] ABSTRACT: Hepatitis E virus (HEV) genotype 3 was first identified in swine raised on a Shanghai suburban pig farm in late 2006. To accurately determine the prevalence of HEV infections among Shanghai pig farms, 426 pig fecal samples were collected from 37 pig farms located in all 10 Shanghai suburban districts and tested for the presence of HEV RNA using RT-PCR. Genetic analysis based on an amplified 150-bp ORF2 fragment revealed 111 samples to be HEV positive, and the prevalence of HEV infection within the different districts varied between 0 and 41.7%. Thirty-two samples were sequenced, and phylogenetic analysis indicated that 10 isolates belonged to HEV genotype 4 and were most closely related to 3 human and 2 swine HEV strains, all of which had originally been isolated from Asian countries including Japan and China. The remaining 22 isolates belonged to genotype 3 and were most closely related to a strain of swine HEV, US-SW, isolated from pigs in the United States. Our data indicated that genotype 3 HEV was widespread among suburban Shanghai pig farms although further study is required to determine the source and zoonotic nature of the virus.
[Show abstract][Hide abstract] ABSTRACT: Strains of hepatitis E virus (HEV) genotypes 1 and 4 have been detected on the Chinese mainland although there have been no previous reports of zoonotic genotype 3 HEV. In the present study, 65 swine fecal specimens were collected from five pig farms located in different Shanghai suburbs. RT-PCR and nested PCR were undertaken using partial nucleotide sequences of Open Reading Frame 2 (ORF2) of HEV to detect HEV RNA. Genetic analysis was based on alignments of an amplified 150-nt ORF2 sequence. RT-PCR revealed 15 HEV positive samples among 65-pig fecal specimens examined. Phylogenetic analysis of the amplified sequences indicated seven HEV strains belonged to genotype 3 and eight strains to genotype 4. This is the first time that genotype 3 hepatitis E virus has been identified on the Chinese mainland.