Development Of National Reference Product Of SFTSV Nucleic Acid Detection Kit And Study On E Protein Of Japanese Encephalitis Virus

Part one:Development of national references for nucleic acid detection kits of Severe Fever with Thrombocytopenia Syndrome bunyavirus [Objective] Severe Fever with Thrombocytopenia Syndrome bunyavirus(SFTSV) was a novel virus that emerged in China for the first time and recovered by domestic experts. There was a high initial case fatality rate of 30%. In view of no effective vaccines or drugs for the virus and in order to control the virus effectively, the diagnosing the disease is of significence. Nowadays there are several kinds of detection kits appeared constantly accompanying, however no relevant standards for SFTSV are available. To control the quality of such detection kits scientifically and effectively, we developed the national references for nucleic acid detection kits of SFTSV. [Methods] According to the relevant guidelines for the development of national references and the biological characteristics of SFTSV, we selected the positive and negative references viruses which met the requirements and similar infection mechnism and clinical symptop. To ensure the accuracy and specificity of the references, we identified them by sequencing and fluorescence quota PCR methods. To study the minimum detection limits of the kits, we used the dilution series of virus HB29 strain and then ensured the sensitivity of the references. The precision of the references was assessed by coefficient of variation(CV) of Ct values tested with virus strain HB29, the concentration of which was 104U/m L. To confirm some specific values of the references, we organized several departments to carry out the collaborative calibration with primary references. The stability of references was also evaluated in different temperatures and repeated freezing and thawing conditions.[Results] We determined 6 positive references and 5 negative references on the basis of broad-spectrum and representativeness. To ensure the accuracy of the references, proof tests by sequencing and fluorescence quota PCR methods were launched later. According to the minimum detection limit test with the dilution series of virus HB29 strain and combined the virus load of clinical patient serum, we confirmed 102U/m L virus concentration as the minimum detection limit. It meant that the content of 102U/m L must be detected as positive and less was not required. With the precision test results of each detection kits, we confirmed that the CV of the precision references’ Ct values should not be more than 5.0%. The collaborative study results by each laboratory were consistent with our results. The results of stability test showed the references owned good stability. Currently the references had been approved by the national standards committee andbeen the first generation national references for nucleic acid detection kits of SFTSV. The approval number was(2015) National Biological Reference 0065.[Conclusion] National references for nucleic acid detection kits of SFTSV were established successfully for the first time. The references filled the vacancy of no relevant references in domestic and played an important role in quality control for the detection kits for SFTSV. This study will promote the prevention and control of SFTSV more scientificly and effectively.Part two:Study on E protein of Japanese Encephalitis virus(JEV)[Objective] Japanese encephalitis live attenuated vaccine SA14-14-2 has been developed by China and was widely used in domestic and abroad and playing a critical role to control Japanese encephalitis epidemic in China and South-eastern Asia. To explore the theoretical basis of stabile attenuated viral virulence and good immunogenicity of the JEV attenuated SA14-14-2 on the molecular and atome level, we analyze the crystal structure of E protein of JEV SA14-14-2 and its previous virus strains SA14-5-3, SA14-9-7 by X-ray diffraction technology accompanying investigation of the biological functions of their E proteins. [Methods] The recombinant plasmid was structured by prokaryotic expression vector p QE C3 and E gene of JEV strains SA14-14-2, SA14-5-3, SA14-9-7, then transformed into recipient bacteria BL21 after verifying the correct construction of plasmid. The target proteins were induced by TPTG and then broken bacteria BL21 by untrasonic to gain the inclusion body of E proteins. By Nickel column affinity chromatography、 folding renaturation and gel filtration chromatography skills, the purified proteins were harvested. Some of the E proteins were applied to crystal structure analysis and the others were used to immunize Balb/c mice with different dosage of 80μg and 120μg per mouse to test the neutralizing antibody and immune protection.[Results] The recombinant vector of p QE C3 and JEV envelope glycoprotein E genes were constructed correctly and were transformed into the expression bacteria BL21 and identified. The target protein inclusion bodies were induced by IPTG and then got the active proteins by renaturation. Finally we gained the E protein of SA14-14-2、SA14-5-3 and SA14-9-7 by purification. The optimal conditions of crystalization of JEV E proteins estabilished, and the crystals of JEV E proteins have been formed. The results of immunogenicity experiments showed that the neutralizing antibody level afterimmunizing of SA14-14-2 E protein was the highest(1:23) and the serological conversion rate reached 100%. The trend of neutralizing antibody level and the serological conversion rate were SA14-14-2>SA14-5-3>SA14-9-7, which was correspond to the immunogenicity trend of this three viruses. These three proteins all exhibited the protective ability to a certain extent, but the difference was not obvious.[Conclusion] The E proteins of JEV were gained through prokaryotic expression and this provided the conditions for the further studies of the function and crystal structure of the proteins. Furthermore, this laid the technical foundation for analyzing the pathogenic mechanism of immunogenicity mechanism of JEV SA14-14-2 vaccine and the quality control of vaccine. The neutralizing antibody level results indicated the immunogenicity of JEV SA14-14-2 E protein was higher than SA14-5-3 and SA14-9-7, which accorded with the immunogenicity ability of these three viruses. This suggested that the E protein was a key protein to the immunogenicity change of JEV SA14-14-2, SA14-5-3, SA14-9-7. Because no obivious difference of the protective ability of three proteins was observed, we infered that the immunogenicity mechanism of JEV SA14-14-2 might be related to other non-structural proteins.