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Investigation on Contamination of Human Parvovirus B19 in Blood Products and Establishment of A New Inactivation Method for the Virus

Author: WangMin
Tutor: HouJiFeng
School: Chinese Pharmaceutical and Biological Products
Course: Immunology
Keywords: Human parvovirus B19 Blood products Real-time PCR UV- inactivated virus UVC Protein biological activity 2-DE HPLC
CLC: R927
Type: Master's thesis
Year: 2011
Downloads: 67
Quote: 0
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Abstract

Human parvovirus B19 (human parvovirus B19, or B19), is the only known human pathogenic parvovirus virus no envelope, heat, diameter small (18 20 nm), the genome is a linear single-stranded DNA molecules about 5.5kb. Universal susceptibility of B19 virus can be spread through the respiratory tract, the late winter and early spring is the peak of infection, almost into a worldwide distribution. Also because of its characteristic of having a smaller diameter and heat, having a higher resistance to virus inactivation and removal method used in the existing blood products Production, the existing production process the production of blood products, there is a certain degree of the B19 virus contamination. risk. Therefore, the strengthening of raw plasma screening to reduce the production of the raw material plasma viral content is one of the important means to ensure the safety of the products. The PCR technique is simple, intuitive, good reproducibility, specificity, accurate quantitative advantages. Therefore, the first part of the research project, the real-time quantitative PCR method for investigation of domestic raw plasma and virus contamination of blood products, blood products, production process improvement and quality control to provide the basis. 2276-plasma donation of Members of plasma samples, 9 initial positive screening positive samples retest 3 times to confirm positive 4 parts. Finished detect blood products, intravenous human immunoglobulin sample of 84 batches were negative; the human fibrinogen samples of 14 batches, a batch of confirmed positive; human coagulation factor VIII sample batch of 25, 9 batch of confirmed positive; human thrombin original composite sample of 24 batches, 18 batches of confirmed positive. The positive results of this study and the results of foreign literature is more consistent, and has laid a good foundation for further study. Traditional blood products, viral inactivation processes such as heating method, S / D, low pH method, preferably lipid-enveloped virus inactivating effect of non-lipid-enveloped virus inactivating effect is poor or ineffective. In recent years, non-lipid-enveloped virus inactivation method continue to emerge, such as nano-membrane filtration, heat-inactivated law, but these poor human parvovirus B19 effect. The second part of this research project is to establish a new shortwave ultraviolet viral inactivation method using UVC the inactivated the instrument UVivatec (developed for the joint development of Germany's Bayer Sartorius) analog viruses in blood products - pig small The inactivated virus (PPV) authentication. Set the UV radiation dose were 200,250 and 300J/m2, using the micro-cytopathic method detects residual virus titer, validated inactivation effect, to obtain satisfactory results. Shortwave ultraviolet UVC method allows the the the PPV virus titer reduce 4log more in line with the requirements for viral inactivation validation guide. The third part of the research topic, shortwave ultraviolet UVC viral inactivation method of human thrombin the original complex products quality. UVC inactivation treatment of the sample before and after the activity was measured and the ratio of the analysis found that in the case of not added any protective agent, the activity of coagulation factor recovery was greater than 70%. Its evaluation to exclude subtle changes of the protein structure, while the use of two-dimensional electrophoresis (2-DE), high performance liquid chromatography (HPLC) technology platform. HPLC analysis confirmed after UVC irradiation, the protein polymer content did not change significantly compared with before irradiation protein chromatographic behavior consistent; tests showed 2-DE map of protein spots almost equal number after the processing of the samples inactivated protein spots basically the same size and position. Human parvovirus B19 universal susceptibility of patients with lower immunity has the potential pathogenic threats, potential danger to the entire plasma pool contaminated plasma donation member containing a single unit of plasma virus shortwave ultraviolet UVC virus-free membrane The inactivated better. To exclude UVC shortwave UV irradiation so that the protein to form a polymer or cleaved into small protein fragments, thereby causing the active function of the change of the effect, we used activity detection method and the 2-D and SEC-HPLC after the inactivation treatment of protein in the sample study the function and structure of various detection methods complement each other to obtain satisfactory results. The activity stability of the samples before and after the UVC inactivated activity recoveries were in the 70% or more, the small difference between the inactivated inactivated before and after different doses and the activity in the reference range of Pharmacopoeia. The shortwave UV prominent non-enveloped virus inactivation advantage, also provides us with new ideas and methods.

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