Development Of An Immunochromatographic Test Strip And Inactivated Vaccine Of Porcine Hemagglutinating Encephalomyelitis

Porcine hemagglutinating encephalomyelitis is an acute, highly contagious disease in piglets caused by the hemagglutinating encephalomyelitis virus (HEV). HEV belongs to the family of Coronaviridae, which included encephalomyelitis and Vomiting and wasting disease clinically type and mainly infects 1-to 3-week-old piglet causes vomiting, exhaustion, and neurological signs. The mortality rate varies from 20% to 100%. In 1962. the pathogen was isolated for the first time from suckling piglets suffering from encephalomyelitis in Canada. In 1972, the PHEV-VW572 strain was isolated from the tonsils of pigs that exhibited only vomiting and exhaustion symptoms, but no neurological signs. Serological surveys revealed that HEV infections in pigs are very common worldwide, most of the infected pigs in a sub-clinical state, once the disease, will cause huge economic losses. At present, various laboratory methods are available for the detection and surveillance of HEV. including virus isolation and identification, RT-PCR, nested PCR, hemagglutination and hemagglutination inhibition (HA and HI,respectively) tests, the enzyme-linked immunosorbent assay (ELISA). and the virus neutralization (VN) test. However, these detection methods are laborious, time-consuming, and require laboratory operations or special equipment, which makes these methods unsuitable for on-site inspection. As such, the current methods would not be useful for managing an outbreak of the disease. Therefore, it was necessary to develop a sensitive, specific, and easily performed detection method for on-site detection of HEV or HEV antibodies in order to increase the surveillance of HEV infections.In recent years, with the monoclonal antibody (McAb) technology development and maturation, molecular biology, immunology, bacteriology, virology, biochemistry, genetics and many other fields have been widely used. Many diseases, diagnosis and differential diagnosis were established with the McAb.In this study, Balb/c mice were immunized subcutaneously with HEV purified by sucrose density gradient centrifugation, their splenic mononuclear cells were isolated and fused with murine myeloma cells (SP2/0). The hybridomas were generated through the selection of HAT medium and screened using Enzyme-linked immunosorbent assay (ELISA) and access to four single-secreting anti-HEV McAb, named 2H2,2A1,1E2,4D4. Identified,4 McAb ELISA titers between 1:12800 and 1:51200; subgroup identification,1E2 for the IgG2a,2H2,2A1 and 4D4 for the IgG1;Western blot analysis showed that 2H2,1E2 and 4D4 to recognize HEV hemagglutinin-esterase protein (HE),2A1 may recognize spike protein (S). Development of the sandwich ELISA with McAb 2A1 and 2H2 for detection HEV, the minimum could be detected 3.75μg/mL for HEV, and infection with the transmissible gastro-enteritis virus (TGEV), porcine Epidemic diarrhea (PEDV), porcine pseudorabies virus (PRV), Hogcholera virus (HCV) and bovine coronavirus (BCV) and so no cross reaction, wihich shows that the sandwich ELISA can be used for HEV detection.Colloidal gold immunochromatographic assay (GICA) is a solid-phase immunoassay developed in the 1980s that combines the techniques of colloidal gold labeling, chromatographic analysis, immunodetection and other methods. Because of GICA's convenience and speed as well as its specificity and sensitivity and ability to be used without instruments or with only a simple instrument, it is suitable for clinical diagnosis and drug testing purposes and can be used anywhere.In this study, an immunochromatographic strip with high sensitivity and specificity was successfully developed for the detection of HEV. combining McAb and GICA.The colloidal gold particles were consistent in size and uniformly distributed, with a mean diameter of about 30nm when observed under a transmission electron microscope; monoclonal antibodies by gradient method to determine the best combination of colloidal gold pH 8.5, the best combination of a concentration of 37μg/mL. The colloidal gold-labeled MAb 2H2 solution was dispensed onto glass fiber paper at a speed of 50μl per cm using an XYZ3000 Dispense Workstation, and the MAb 2A1 or the goat anti-mouse antibody was dispensed at the test or the control line on the NC membrane using XYZ-3000; the sample pad, pretreated conjugate pad. NC membrane, and absorbent pads were glued together on a support board and assembled into a test strip.The immunochromatographic strip was capable of specifically detecting HEV with 30μg/mL within 10 min. Storage of the strips at room temperature for 6 months or at 4℃for 12 months did not change their sensitivity or specificity. Using RT-PCR and ELISA as reference test, the excellent agreement (98%; kappa= 0.956) between the results obtained by RT-PCR or ELISA and the immunochromatographic strips. Based on its high specificity, sensitivity, and stability, the immunochromatographic strip would be suitable for on-site detection of HEV for surveillance and epidemiological purposes.An immunochromatographic strip was developed for the detection of an antibody against HEV. Colloidal gold-labeled rabbit anti-pig immunoglobulin G (IgG) was used as the detection reagent, and the HEV recombinant antigens and goat anti-rabbit IgG were coated on the prototype strip and the control lines, respectively. The immunochromatographic strip was capable of specifically detecting HEV antibodies in serum with a HI titer of 2 within 10 min. Storage of the strips at room temperature for 6 months or at 4℃for 12 months did not change their sensitivity and specificity. Using HI as a reference test, there was a strong agreement between the results obtained by HI and the immunochromatographic strips (kappa=0.911). Additionally, there was a strong agreement between ELISA and immunochromatographic strips (kappa=0.953). When the immunochromatographic strip was used for serological diagnosis of 672 serum samples in the Jilin and Liaoning province in China, the seropositivity ranged from 42.08% in Jinzhou District to 65.33% in Baishan District. Based on the high specificity, sensitivity, and stability of the immunochromatographic strip, it would be suitable for on-site detection of HEV antibodies in order to monitor HEV infections in an animal population.Currently, there is no effect on the prevention or treatment of HEV drugs or vaccines has been reported, applied only in some countries abroad, "subclinical infection" of prevention, that is, one month before labor in sows contact with sick, spray or intramuscular injection training the attenuated HEV. When the sow immunitied, piglets gain the antibodies through colostrum, but it does not show clinical symptoms, most of these pigs showed a subclinical infection, and this immunological method exist clinical infection hidden danger. Therefore, the development of efficiently and safely vaccine against HEV prevention has important practical significance. In this study, based on HEV-67N strain as a vaccine candidate strains developed by optimizing the in vitro culture conditions to determine the HEV-67N PK-15 cells in culture and passage, using the nutrient medium with 0.4% BSA; establishment of PK-15 cells of the original seed cell banks (123～126 cells), based on the seed cell bank (127～136 cells) and the working seed cell bank (137～141 cells); the same time establish HEV-67N, respectively, the original seed virus database. foundation seed and working seed virus database virus database; random sample of the virus in the cell library and PK-15 cells and HEV-67N by microscopy, sterility test, mycoplasma detection and analysis of nucleic acids which adhere to "quality standards for veterinary biologics." In the adjuvant selected on the basis of quality, taking the virus working seed virus database solution, inactivated by 4‰formaldehyde, By "People's Republic of Veterinary Biological Products Quality Standards " provides, the aluminum hydroxide gel adjuvant and inactivated virus at 1:9 ratio of mixing, preparation HEV cell culture inactivated vaccine. Randomly selected the vaccine which prepared in labs Vaccination to Balb/cmice and pregnant sows, the results show that favourable specific immune response induced by the vaccine. The vaccine is also safe an effective to mice and pregnant sows. There is no adverse reaction happened on mice and sows after injecting the vaccine. On post-vaccination antibody titers have different Balb/c mice brain attack drugs, the protection rate is more than 80% when the mice's HI antibody titers≥2(?).There is satisfactory parallel relationship between HI antibody titers and immune attack drug test. It can replace convetional attack protection rate test. HEV inactivated vaccine inoculated Balb/c mice, HI antibody titers of mice up to 29 after the third immunization. The immune mice could produce specific humoral immune response certificated by serum sub-categories, lymphocyte proliferation, cytokines and other methods. The main of immune response is Th2 type. Finally, the preservation of the vaccines evaluated. The physical properties and immune effect of the vaccine was no change when the vaccine stored at 2-8℃for 12 months and room temperature (13℃-28℃) for 4 months.It basically the same characteristics as inactivated vaccine stability, ease of transportation and conservation. Thus, in this study, the body could produce specific immune response by the prepared HEV vaccine safely and efectively. Piglets generated the antibodies from the immunization of pregnant sows to achieve the purpose of preventing the disease.The vaccine has important practical significance to prevention of HEV...