Analysis Of S Region Gene Mutations In Occult Hepatitis B Virus Infection

Objective:This study was to evaluate the detection performance of CIA by analyzing the expression patterns of serological markers of clinically HBsAg-negative and HBV DNA-positive HBV occult infections.The amino acid substitution caused by the mutation of the HBV S region gene was analyzed by sequencing the HBV S region gene of the OBI specimen,especially the nucleotide in the"α"determinant.Further analysis of high frequency mutation sites and epidemiological data of HBV S region in this region,to explore the causes of OBI and its pathophysiological mechanism.Methods:This study was divided into two parts.The first part of the study:1527 serum samples from clinically HBcAb-positive and HBsAg-negative serum samples which divided into five groups were collected.HBsAb with OD450nm value<0.070 were selected by ELISA screening HBcAb,and HBV DNA was detected by high-sensitivity PCR detection kit.The second part of the study:To collect the first part of the study was tested by HBV DNA test positive OBI group and HBV DNA high load chronic hepatitis B patient control sample.The HBV S region of the full-length DNA in the OBI clinical sample was amplified by two rounds of PCR amplification technology.The nucleotide sequence of the HBV S region gene was analyzed by direct sequencing or cloning sequencing of the PCR product,and the obtained S region gene sequence was compared with the reference sequence of the corresponding gene subtype to obtain the HBV S region gene mutation of each sample and amino acid variation.Statistical analysis:SPSS19.0 statistical software was used for data processing and statistical analysis.Theχ~2 test was used to compare the rates of multiple groups and groups.When P<0.05(bilateral)was used,the difference was statistically significant.(In the comparison of the rate of each group,when the sample content is>40 and the theoretical frequency in each grid is≥5,theχ~2 test is used;when the sample size is>40but 1≤theoretical frequency is<5,the chi-square test is required correction formula)Result:The first part of the study:1527 HBcAb-positive and HBsAg-negative serum samples were screened HBcAb by ELISA with OD 450nm value<0.070 423 cases,423serum samples were tested by high-sensitivity PCR,and 42 cases of HBV DNA were positive(HBV DNA>20IU/ml was positive).The rate is 2.75%(42/1527).28 cases of HBV DNA load were 20~1×10~2IU/ml;8 cases were 1×10~2~1×10~3IU/ml;6cases>10~3IU/ml,mainly in low level.The HBV DNA detection rate was relatively high in HBeAb and HBcAb positive group,and HBsAb,HBeAb and HBcAb positive group.The positive rate of HBV DNA in HBcAb S/Co≥10.0 samples was significantly higher than that in other serological model groups,reaching 5.26%(4/76).The second part of the study:Only 20 of the 42 samples in the OBI group successfully amplified bright target gene bands,and 100 cloned strains were sequenced and analyzed.Each sample had 5 clones,and 68 cloned plasmids were confirmed to be HBV S regin gene by sequencing.In the chronic hepatitis B control group,all of the 10samples amplified a bright target gene band,and the PCR purified products were sequenced into the HBV S region gene.There were 62 mutations in 16 sites in the MHR region of OBI group,the mutation rate was 3.80%(62/1632),among which the common mutation sites in the“α”determinant were:I126T,C124R,M133L;common in MHR region.The mutation site has(excluding the"α"determinant):K122E,L162R,V106G.A total of 5 mutations occurred in the MHR region of the chronic hepatitis B control group,4 times in the"α"determined cluster.The mutation rate of the OBI group in the MHR region was significantly higher than that in the chronic hepatitis B control group(P<0.05).The main mutation types of HBV type C such as I126T,K122E and L162R found in this study are rarely reported in the literature.Conclusion:As a method for detecting sensitivity and specificity of HBsAg highly in the industry,the CIA method still has the phenomenon of missed detection of HBsAg.Clinically,patients with HBsAg-negative high-reactivity HBcAb(S/Co value≥10.0)should be further tested for HBV DNA to confirm the presence of OBI.Each blood collection center should expand the use of NAT technology,and the HBcAb project can be appropriately added as a blood donation screening project to prevent blood products which could flow to the clinic to cause hematogenous HBV infection from OBI blood donors.It is necessary to develop new serological diagnostic reagents for hot spot mutation sites in China,enhance the sensitivity and specificity of HBsAg detection reagents,and improve the ability to identify variant HBsAg.