Epidemiology Of HCV Genotypes And Subtypes In Yunnan And Establishment Of Detection Method For HCV Resistance Mutations Using NGS Technology

Hepatitis C is a major blood-borne disease caused by hepatitis C virus(HCV).Hepatitis C virus is a global epidemic.According to statistics,there are about 185 million people infected with HCV worldwide.China is a highly infected area of HCV,and the number of reports of hepatitis C is also on the rise in viral hepatitis.The genotype distribution of HCV is complex and shows significant regional differences.At present,HCV has found more than 80 subtypes of 8 genotypes.In China,types 1b and 2a are the most common.At present,direct antiviral drugs(DAA)against HCV have been used clinically,which has potent inhibition of viral replication,rapid reduction of HCV viral load,and improved sustained viral response(SVR)for hepatitis C treatment.However,with the widespread use of DAA drugs,coupled with the high mismatch rate of HCV virus replication and the low fidelity of RNA polymerase,HCV produces mutations in drug resistance genes under the pressure of DAA drugs,which in turn affects hepatitis C.treatment effect.Mutations in HCV-related resistance genes are directly related to different genotypes and subtypes of HCV.Therefore,elucidation of the distribution of HCV genotypes and subtypes and the characteristics of drugresistant mutant genes have important guiding significance for the clinical treatment of hepatitis C.In addition,the detection of HCV DAA-resistant mutations is mainly through the first-generation sequencing(Sanger)method,which has low sensitivity and small amount of information,which also restricts the monitoring of HCV resistance sites.Based on this,this paper studied the epidemiological characteristics of HCV genotypes and subtypes in Yunnan,and established a detection method based on the next generation sequencing technology(NGS)HCV resistant mutant gene.First,this paper studied 1464 samples from Yunnan in 2018.The results showed that HCV genotypes were 3b(39.3%),1b(21.0%),3a(20.0%),6n(8.8%),2a(7.2%).6a(2.1%),showing the distribution characteristics of 3b,3a and 1b.In addition,we analyzed the genotype dynamics of 503 HCV-positive samples in Kunming from 2008 to 2018.The results showed that the overall HCV genotype distribution was 3b(37.6%),3a(21.9%),and 1b(19.3%).),2a(10.5%),6n(7.2%).1b from the 13% in 2008 to 4% in 2018,2a from 10% in 2008 to 0% in 2018 showed a significant downward trend year by year.On the contrary,3a from the 3% in 2008 to 16% in 2018,3b from 9% in 2008 to 24% in 2018 showed a significant upward trend year by year.Secondly,in order to investigate the prevalence of drug-resistant mutations in HCV treatment in Yunnan,we randomly selected the main epidemic HCV 1b(60 cases),3a(60 cases),3b(60 cases),2a(30 cases),6n(40).example).The target genes NS3,NS5 A and NS5 B of DAA were sequenced by RT-PCR.The results showed that the NS3 region of HCV 1b was prone to M/V170I(54.5%)versus boceprevir,M/V170I(52.7%)versus cimetime,M/V170I(52.7%)versus telaprevir,M /V170I(52.7%)resistance to resistance to vezivirin;L28M(42.6%)is prone to occur in NS5 A region and resistant to resistance to dacaseta,radipavir,and olviralvir;NS5B is easy C/R316N(80.4%)occurred and was resistant to sispervir.The NS3 region of 3a is prone to H170I(98.2%)and is resistant to resistance to cevirix.The NS3 region of 3b is prone to H170I(98.1%)and resistant to resistance to cecilyvir;the NS5 A region is prone to Q30K(100%)and resistant to erbavir,radipavir,and verapamil.Sexually resistant,D/G31M(98.1%)and resistant to resistance to dartarevir and erbavir.Finally,we designed 50 pairs of multiple HCV NS3,NS5 A and NS5 B drug loci based on the distribution of six major epidemic subtypes of HCV in Yunnan(1b,2a,3a,3b and 6n).PCR primers were used for template amplification.The results of NGS assay showed that high-throughput sequencing could detect low-frequency resistant mutation sites that were not detected by Sanger sequencing.The results of the two other detection methods were basically the same and the mutation frequency detected by NGS sequencing.It is 100%.In addition,we also carried out specific validation.The results indicated that the multiplex PCR primers designed in this method are specific to the HCV virus genome,indicating the initial establishment of the next-generation sequencing method for HCV-resistant mutant genes.In summary,this paper clarifies the distribution characteristics of current HCV genotypes and subtypes in Yunnan,and the changes of HCV genotypes and subtypes in Kunming for 11 years,providing a scientific basis for the treatment and prevention of HCV patients.In addition,we first studied the prevalence of HCV major prevalence and subtype pre-existing drug resistance mutations in DAA before treatment in Yunnan,and initially established a detection method for HCV different genotype subtype resistance mutation genes based on NGS technology,which is the next step for accurate monitoring.Drug-resistant mutations in the treatment of HCV DAA provide important technical means.