Immunization against porcine epidemic diarrhea virus and vaccine development / 生物工程学报

Porcine epidemic diarrhea (PED) is a major disease of pigs that inflicts heavy losses on the global pig industry. The etiologic agent is the porcine epidemic diarrhea virus (PEDV), which is assigned to the genus Alphacoronavirus in the family Coronaviridae. This review consists of five parts, the first of which provides a brief introduction to PEDV and its epidemiology. Part two outlines the passive immunity in new born piglets and the important role of colostrum, while the third part summarizes the characteristics of the immune systems of pregnant sows, discusses the concept of the "gut-mammary gland-secretory IgA(sIgA) axis" and the possible underpinning mechanisms, and proposes issues to be addressed when designing a PEDV live vaccine. The final two parts summarizes the advances in the R&D of PEDV vaccines and prospects future perspectives on prevention and control of PEDV, respectively.

The 40-91 aa sequence of porcine epidemic diarrhea virus ORF3 protein is the key structural domain controlling its location in cytoplasm / 生物工程学报

ORF3 protein, the single accessory protein encoded by porcine epidemic diarrhea virus (PEDV), is related to viral pathogenicity. In order to determine the cytoplasmic location signal of PEDV ORF3, we constructed a series of recombinant plasmids carrying full-length or truncated segments of PEDV DR13 ORF3 protein. When the acquired plasmids were transfected into Vero cells, expression and distribution of the EGFP-fused full-length ORF3 protein and its truncated forms in the cells were observed by laser confocal microscopy. The results showed that ORF3 protein or their truncated forms containing 40-91 aa segment including two transmembrane domains were localized in the cytoplasm, whereas ORF3 truncated peptides without the 40-91 aa segment were distributed in the whole cell (in both cytoplasm and nucleus). This suggests that the 40-91 aa is the key structural domain determining cytoplasmic location of PEDV ORF3 protein. The discovery provides reference for further clarifying intracellular transport and biological function of PEDV ORF3 protein.

Establishment and application of visual LAMP detection method of infectious bovine rhinotracheitis virus / 生物工程学报

Three pairs of primers were designed according to the conserved region of IBRV gB gene published in GenBank(GenBank Accession No. DQ006857.1) using the software Primer Explorer V4. The loop mediated isothermal amplification (LAMP) assay was established by optimization of the reaction system and then evaluated through sensitivity and specificity tests. In total 393 clinical specimens were detected for IBRV using the established LAMP assay performed at 65℃ for 50 min, which produced a ladder-like pattern of amplification bands and the detection result could be judged by color change. The sensitivity of the assay was 10 copies/μL plasmid DNA which was 1000 times higher than that by PCR method and equivalent to nested-PCR. There was no cross-reactivity of the assay with bovine viral diarrhea virus (BVDV), pseudorabies virus (PRV) and vesicular stomatitis virus (VSV). The positive rate of 301 nasal swabs and 92 serum specimens were 87.6% and 58.8%, respectively, which meant nasal swab specimen was more suitable for clinical IBRV detection by the method. The IBRV LAMP method established in this study has the advantages of visualization, quickness, specificity and sensitivity and be suitable for rapid detection of clinical IBRV detection on the spot.

Advances in reverse genetics to treat porcine epidemic diarrhea virus / 生物工程学报

Porcine epidemic diarrhea virus (PEDV) is one of the major etiologies responsible for the acute, highly contagious disease in the digestive tract of pigs, especially neonatal piglets. Since PEDV was first identified in Europe in the late 1970s, it has resulted in significant economic losses in many Asian swine-raising countries, including China. Recently, reverse genetics techniques including targeted RNA recombination, bacteria artificial chromosome system and in vitro ligation have been successfully used to manipulate the genome of PEDV, which providing new strategies for the clear delineation of the functions of the viral proteins, the mechanisms behind PEDV pathogenesis and the design of novel vaccines against PEDV. Here, we review the progresses of different reverse genetics platforms developed for PEDV and their applications, covering the roles of trypsin in PEDV propagation, functions of S and ORF3 protein and the development of next generation PED vaccines, and the perspectives of reverse genetics for PEDV.

Effects of dietary fiber and inositol hexaphosphate on the onset of carcinoma of large intestine in rats induced by 1,2-dimethylhydrazine / 中国组织工程研究

BACKGROUND: Incidence rate of carcinoma of large intestine increases with the development of national living standard. Many epidemiologic surveys have showed that dietary factors have closely correlation with high risk of carcinoma of large intestine.OBJECTIVE: To observe the effect of dietary fiber and inositol hexaphosphate on the incidence of carcinoma of large intestine by establishing rat models with carcinoma of large intestine.DESIGN: Randomized block design.SETTING: Institute of Nutriology, Medical College, Qingdao University.MATERIALS: The experiment was performed at the Institute of Nutriology, Medical College, Qingdao University from March to December 2004.Totally 86 male Wistar rats of 4 weeks were randomly assigned based on body mass into cellulose group with 14 rats, pectin group with 14 rats, inositol hexaphosphate group with 15 rats, cellulose plus inositol hexaphosphate group with 14 rats, pectin plus inositol hexaphosphate group with 14rats and control group with 15 rats.METHODS: Basic feed without dietary fiber was given in the control group. 10% pectin was added in the pectin group. 10% cellulose was added in the cellulose group. 2% sodium phytate was added in the water of inositol hexaphosphate group. 10% pectin and 2% sodium phytate were added in the water of pectin plus inositol hexaphosphate group. 10% cellulose and 2% sodium phytate drinking water were added in the cellulose plus inositol hexaphosphate group. A total of 86 rats received 1,2-dimethylhydrazine (DMH) by subcutaneous injection to induce carcinoma of large intestine at week 4 after breeding. Incidence rate of large bowel neoplasm,number and volume of tumor were observed. Proliferating activity of large intestinal mucosa cells was determined (the ratio of number of positive cells of proliferating cell nuclear antigen to total count of nucleus).MAIN OUTCOME MEASURES: ①Incidence rate of large bowel neoplasm in each group, changes of mean number and mean volume of tumors of each rat, and ②proliferating activity of large intestinal mucosa cells of rats.RESULTS: ①Most of the rats in each group died during the 20-week trial. Oue rat died in the pectin group, the pectin plus inositol hexaphosphate group and the control group, respectively after 20 weeks. ②There was no significant difference in the incidence rate of large bowel neoplasm of rats compared with the control group (P ＞ 0.05), but the mean number and mean volume of tumors of each rat in the inositol hexaphosphate group were significantly lower than those in the control group [quantity: (1.1±0.2),(4.1±1.2)rats,P ＜ 0.01 ;volume: (176.1±65.5), (1 046.7±469.0)mm3,P ＜ 0.05], and the mean number of tumors of each rat in the pectin plus inositol hexaphosphate group was obviously higher than that in the control group [(7.5±1.9), (7.2±1.0) rats,P ＜ 0.05]. ③The proliferating activity of large intestinal mucosa cells in the inositol hexaphosphate group significantly decreased as compared with the control group. The proliferating activity of large intestinal mucosa cells in the pectin group increased as compared with the control group [(41.8±4.7)%, (83.6±2.9)%,(66.7±7.8)% ,P ＜ 0.01 and 0.05].CONCLUSION: It may increase the incidence rate of carcinoma of large intestine when pectin is added in diet, on the contrary, the rate decreases if inositol hexaphophate is added in the water.