The Prevalence Of HBV Among Ethnic Minorities In Yunnan And The Establishment And Evaluation Of HBV PgRNA Detection Methods

Hepatitis B virus(HBV)is a small circular partial double stranded(ds)DNA virus with an envelope,belonging to the heparophilic DNA virus family.HBV infection is a major pathogenic factor leading to chronic hepatitis b(CHB),liver failure,cirrhosis and hepatocellular carcinoma(HCC),posing a great threat to human health.Due to the differences in pathogenicity and therapeutic effects of different genotypes and subtypes on patients with hepatitis b,yunnan has a unique geographical location and the regional characteristics of multi-ethnic clusters.Therefore,it is of great significance for the treatment and control of hepatitis b to carry out molecular epidemiological studies on HBV in minority populations in yunnan.In addition,recent studies have found that HBV Pg RNA is a direct transcription product of HBV ccc DNA,which can reflect the progress of chronic hepatitis b by reflecting the transcription activity of HBV ccc DNA and thus guide clinical treatment and prognosis.However,currently there are few studies on detection methods for HBV pg RNA and this method has not been widely used in clinical practice.Based on this paper,the general population of eight ethnic minorities in yunnan province was investigated for HBV epidemiology,and the HBV pg RNA detection method and its evaluation were established.First of all,according to the general population we collected five ethnic minorities in yunnan region(wa,the blang nationality,hani,naxi,pumi)for a total of 2696 cases of plasma samples,using the method of Elisa detection found that HBs Ag total positive rate was 7.6%(204/2696),including pumi infection rate was 15.4%(58/376),7.6%(37/487)of the hani nationality,wa 7.4%(66/896),5.4%(25/464)of the blang nationality,the naxi 4.9%(23/473).Genotypes and subtypes of 204 HBs Ag positive samples were analyzed using HBV S gene.The results showed that HBV subtypes in this population were B2(48.6%),C1(34.2%),B4(13.9%),C2(0.7%),C5(0.7%),and 4.9%(10 cases).In order to further compare HBV subtypes distribution differences in different ethnic minorities,we collected 206 cases also include three national(dai,yi nationality and han nationality),blood samples,hepatitis b liver disease by gene subtype analysis shows that the crowd HBV subtypes distribution for B2(29.6%),C1(29.1%),C2(23.3%)and B1(0.5%),B4(0.5%)and 35 classification samples yet.By statistical analysis found that hani is given priority to with B2(75%),pumi to B2(38.6%)and C1 subtype(43.9%),the naxi is given priority to with C1 subtype(47.1%),yi to genotype B2(32.69%)and genotype of C1(38.46%)subtype is given priority to,the dai is given priority to with genotypes C1(67.35%),the han nationality in genotype B2(30.48%)and C2(36.19%).Secondly,in order to further analyze the genetic characteristics of the above 45 untyped samples,we amplified the whole HBV genome sequence,among which we successfully obtained the whole HBV gene sequence of 39 cases.The sequence analysis found that 8 cases belonged to B/C recombination and 1 case belonged to B/I recombination.Seven patients were of the potential new subtype B,six were of the new type C,and the remaining 16 were of different subtypes.To further naming these new HBV subtypes,based on HBV genome sequences we separately analyzed HBV genetic distance,homologous relationship between evolutionary tree analysis,gene recombination,as well as the specific amino acid mutation analysis,the analysis found the samples in line with the HBV gene subtype naming rules,so we named them B10 and C17 subtypes.Finally,according to the characteristics of HBV Pg RNA5’and 3’end sequences,we established two kinds of HBV Pg RNA fluorescence quantitative PCR detection methods,respectively.After specificity,sensitivity and repeatability analysis,it was found that the detection method for HBV5’end sequences was more specific and more sensitive.In addition,we selected 40 cases of clinical sample can we build the HBV Pg RNA HBV test method of evaluation,the results show that the HBV Pg RNA content in the samples of chronic hepatitis b is significantly higher than hepatitis b liver samples,the results are consistent with clinical symptoms consistent with literature reports,the results show that the method we established HBV Pg RNA has potential clinical application value.To sum up,this study illustrated the distribution of HBV genotypes and subtypes in eight ethnic groups in yunnan province and compared the differences in genotypes among different ethnic groups,revealing the complexity and diversity of HBV genotypes among different ethnic groups in yunnan province.At the same time,we also established and evaluated two kinds of detection methods of HBV pg RNA fluorescence quantification,which provided an important theoretical basis for the future research of HBV and a more convenient and sensitive detection method for the diagnosis of clinical samples.