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To establish a serum HCV genotyping methods and the preliminary clinical evaluation

Author: YangXiQin
Tutor: PengRuiYun; ZhangHeQiu
School: PLA Military Academy of Medical Sciences
Course: Pathology and Pathophysiology
Keywords: Hepatitis C virus Competitive inhibition ELISA Genotyping Serotyping
CLC: R373.21
Type: Master's thesis
Year: 2013
Downloads: 13
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Abstract


It is approximately180million patients (3%of total population in the world)infected by HCV and130millionpatients are chronic HCV based on the statisticsfrom World Health Organization (WHO). According to the research result from thesecond serum epidemic of viral hepatitis between1992and1995, the average positiverate of anti-HCV by Chinese is3.2%, which belonged to moderate epidemic. HCV iseasy for evolving into chronic disease, and about75%to85%for acute hepatitis islikely to develop into chronic hepatitis, even hepatocirrhosis and liver cancer. Theincidence rate of in liver cirrhosis is25%in20to30years by the HCV patients. Theincidence rate of hepatic decompensation and liver cancer are30%and1%to3%peryear in ten years later by the patients of hepatocirrhosis with HCV caused. Insummary, it is bringing massive amounts of damage to the career of public health inChina.HCV is a positive-strand RNA virus that belonged to Flaviviridae, clonedsuccessfully for the first time in1989. Full-length of genome is9.5kb, including acomposed of9033nucleotides with an open reading frame, encoding structuralprotein and non structural (NS) protein. Structural proteins include core protein (C)、envelope protein gp33(E1) and gp70(E2). NS protein includes p7, NS2, NS3, NS4A,NS4B, NS5A, NS5B, etc. Because RNA enzyme lacks proofreading function, thewhole genome presents a high degree of variability. Currently, HCV is divided into atleast six genotypes and over80kinds of subtypes. In China, the main genotypes are1b、2a, and also have other rare genotypes.According to China Guidelines for the Prevention and Treatment of Hepatitis C,the standard therapy proposal of chronic HCV is the combination therapy of pegylatedinterferon alpha and ribavirin. Meanwhile, the genotype is an important baselinepredictor index in the combination therapy proposal, and has an important guidingmeaning for the dose and time of clinical medication.Currently, analysis methods ofgenotype mainly include directly sequenced analysis in a pair of primers, analysis ofrestriction fragment length polymorphism (RFLP) enzyme digestion, method ofspecific probe PCR and gene chip, etc. In spite with high specificity and sensitivity,all analysis methods of genotype require HCV RNA extraction process, which is needto complex manipulation and high technique. Especially, when the HCV-RNA isnegative or destruction of HCV-RNA owing to improper preservation, the sample cannot be genotyped. Therefore, academicians put forward HCV serological typing,which is typed by detecting the genotype-specific antibodies based on HCV genotypespecific epitope.At present, there are three types of international commercialized HCV serotyping reagents, namely Murex HCV1-6type, Chiron RIBA1-3type and Japanese HCV1,2serotyping reagents kits. The Murex HCV1-6type made by an American companynamed Abbott adopts competitive ELISA method, on the basis of1-6type-specficHCV-NS4peptides to identify different serotypes corresponding to the differentgenotypes. Due to the influence of different countries and regions, as well as differentspecimens, the results of reagents for clinical evaluation are not the same.Thesensitivity is around60%-80%, and the genotyping accordance is around90%-97%;Chiron serotyping strip immunoblot assay (SIA) takes NS4zone specific-epitopepolypeptide as the principle, Core zone specific-epitope polypeptide as thecomplement to conduct serological typing. The sensitivity could reach more than80%,the specificity could stay at90%-96%. Besides, the Immucheck HCV Gr assay,though NS4specific-peptidecan determine only for genotypes1,2, the sensitivity is89.6%, the specificity is96.0%, and the accordance to genotyping is86%. However,these commercialized serotyping assays have not been registered in China, its price ishigh and hard to obtain. Therefore, this research is focus on developing a method forserotyping related to the popular genotypes in China, with the purpose of filling inblanks in this area.According to the report, the amino acid sequences of HCV NS4and Core regionwere downloaded from HCV databases(http://hcv.lanl.gov/content/index), andBiosun bioinformatics software was used to predict the epitope of HCV sequences.HCV-NS4epitopes are mainly located in1693-1708aa, and Core epitope is located in67-81aa. Through the HCV databases online for homology analysis, the NS4region ofserotyping I and Ⅱ amino acid sequence are AⅡPDREVLYQEFDEM andVVAPDKEVLYEAFDEM respectively, and Core region of serotyping I and Ⅱ aminoacid sequence is KARRPEGRTWAQPGY and KDRRTTGKSWGRPGY. Then thecorresponding nucleotide sequences of serotyping epitopes were inferred according tothe preferred codons of Escherichia coli. The overlap extension PCR was used to thesynthesis of C-Ⅰ, C-Ⅱ, NS4-Ⅰ, NS4-Ⅱ, C+NS4-Ⅰ and C+NS4-Ⅱ serotyping chimericgene. Afer enzyme digestion and inserted into vector PGEX-4T-2, the C-Ⅰ, C-Ⅱ,NS4-Ⅰ, NS4-Ⅱ, C+NS4-Ⅰ and C+NS4-Ⅱ plasmid were constructed. The6corresponding antigens were obtained with95%purity using GST sepharose FFcolumn for purification.By the indirect ELISA method, the activities of6antigens were detected with50serums of HCV patients. The reaction of three I type antigens of C-Ⅰ, NS4-Ⅰ, chimericC+NS4-Ⅰ are25.71%、74.28%、88.57%with HCV-1b serum, and the cross-reactivityof three I type antigens are20%、40%、66.67%with HCV-2a serum, the reaction of Itype antigens with1b serum are higher than that with2a serum, and shows Iserotype-specific. The reaction of three Ⅱ type antigens of C-Ⅱ, NS4-Ⅱ, and C+NS4- Ⅱchimeric are33.33%、66.67%、73.33%with HCV-2a serum, and the ross-reactivityare11.42%、48.57%、28.57%with HCV-1b serum, the reaction of Ⅱ type antigenswith HCV-2a serum is significantly higher than that with HCV1b, presents Ⅱserotype-specific.In order to eliminate cross-reactivity between type-specific antigen and, serotypeI, Ⅱ serum, this study adopts competitive method of ELISA. After20different sets ofexperiments, the process condition of HCV serotype detection technology are PH9.6carbonate buffer as a coating solution;20%goat serum-0.01M PBS PH7.4as a closedliquid; LD5-HRP as enzyme conjugates (LD5is a kind of new molecules with moreextensive IgG binding characteristics); determining the concentration of a competitiveantigen is1.5μg/μl; a competitive antigen diluent is goat serum (5%), casein (0.5%)-0.01M PBS PH7.4; four NS4-Ⅰ, C-Ⅰ, NS4-Ⅱ and C-Ⅱ type fragment antigen ascoated antigen, two type I, Ⅱ chimeric antigen as the competitive antigen, calculationmethod to interpret the results.128cases for HCV-1b serums and72cases for HCV-2a serums were used toimplement the preliminary clinical evaluation for this technology.102cases for type I,58cases for type Ⅱ, and2cases for type I with type Ⅱ were testing out. Theclassification efficiency is81.00%in genotyping serum. The accordance togenotyping is97.05%(kappa=0.959,p=0.024), which is close to the internationalreport. Through the further research, there is no obvious cross-reactivity with117patients without HCV infection. The classification efficiency is irrelevant to age ofpatients, antibody S/Co and HCV RNA.The points of innovation for this study include two sections type: combination ofCore and NS4regions were used for HCVserotype to overcoming problem of singleantibody spectrum and raising the rate of sample classification. Besides, this studyintroduces a competitive ELISA technique to do the research for HCV serotype andprocess optimization. It is greatly improving the accuracy rate of HCV serotype.This study established a HCV serotype method on the basis of a competitiveELISA technique, which is a good consistency with genotyping technology. Themethod is simple manipulation, rapid, less pollution, and also does not need specialfacilities and professional staffs, especially suitable for promotion by the Chinafundamental Medical institutions, and having a good prospect for clinical application.

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Human Virology ( pathogenic virus) > Enteroviruses and hepatitis virus > Infectious hepatitis
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