Comparison Of Characteristics Of Pre-existing Drug Resistance And Induced Drug Resistance In Patients With Chronic HBV Infection And The Effect Of Rt181 Mutation On S Gene Sequence

Hepatitis B virus(HBV)is a non-cytotoxic hepatotropic DNA virus.HBV infection can lead to hepatitis,cirrhosis and even liver cancer.About 650,000 people die each year from liver failure,cirrhosis and hepatocellular carcinoma(HCC)caused by HBV infection.HBV infection has become an important global health and safety issue.Antiviral therapy is particularly important for chronic HBV infection.To date,anti-hepatitis B virus drugs include two major categories,one is a limited course of immunomodulatory interferon alpha(including common interferon and peginterferon alfa),and the other is no fixed treatment.Nucleoside/nucleotide analogues(NAS)with direct inhibition of HBV DNA polymerase,including lamivudine(LAM),adefovir dipivoxil(ADV),Telbivudine(LDT),entecavir(ETV)and tenofovir disoproxil fumarate(TDF),The antiviral efficacy of nucleoside(acid)drugs has been recognized.However,virus resistance mutation is also an unavoidable problem in the application of nucleoside drugs.Once the virus resistance mutation occurs,it means that the efficacy of the antiviral drug is reduced or invalid,and the patient's condition may further deteriorate.Due to the highly efficient replication of the HBV DNA polymerase encoded by the HBV-encoding reverse transcriptase region,HBV reverse transcriptase lacks proofreading activity,and due to its lack of 3'-5' exonuclease activity,mRNA is intermediate during replication.When reverse transcription is replicated,base pairing errors are prone to occur.This mismatch rate is much higher than other DNA viruses,which leads to the high mutation of HBV gene,which leads to drug resistance,which may be the cause of pre-existing drug resistance.The question is whether the viral resistance mutations we encounter in our clinic are pre-existing or drug-induced?Since there is the possibility of pre-existing drug resistance,is it necessary to detect HBV gene resistance mutation before the application of nucleoside drugs?Is there any difference between pre-existing drug resistance mutations and drug-induced drug resistance mutations?The resistance mutation is usually located in the reverse transcriptase region(RT)region of the HBV polymerase region,while the HBV S gene encoding the surface antigen(ntl 55-835)completely overlaps with its reverse transcriptase RT(131-1161),the two are translated in the same direction.Therefore,when certain bases in the RT region are changed,it will inevitably lead to changes in the corresponding sites of the S gene.The 181th amino acid variation in the reverse transcriptase region not only makes HBV resistant to adefovir dipivoxil,but also has different drug susceptibility to lamivudine,entecavir,telbivudine,and tenofovir.The degree is reduced,therefore,rt181 is a cross-resistant site for nucleoside and nucleotide drugs.This site variation can cause changes in the S gene.Studies have shown that the variation of HBV rtA181T can lead to the S gene of its corresponding site from TGG to TAG,TAG is the stop codon,no longer encodes the protein,resulting in the change of sW172*of S antigen,resulting in the expression of 55 amino acids of S antigen.Truncated,this variant is also known as the rtA181T/sW172*variant,which affects the biological properties of HBV and its pathogenicity.This truncated viral protein usually has a secretory disorder,is blocked in the liver cells,cannot be secreted into the blood,and the serum HBsAg content is reduced.In addition,in addition to the change of sW172*in S antigen,RtA181T mutation can also lead to the change of sW172L in S antigen.The viral protein produced by this mutant also has secretion disorder,and the HBsAg measured in serum also decreases.Studies have shown that rtAl81T/SW172*mutation can induce the occurrence of liver cancer.From the current research results,rt181 mutation may cause a variety of nucleotide drug resistance or sensitivity decline,may also affect the expression of surface antigens and monitoring of HBV infection,and even more may lead to he occurrence of liver cancer.However,we have observed clinically that rtA181 mutation is not only present in patients with liver cancer,but also in patients with chronic hepatitis B.Therefore,this study aims to explore the presence of pre-existing drug resistance in people with chronic HBV infection.What is the pattern of pre-existing resistance?Are pre-existing drug-resistant variants different from antiviral-induced resistance variants?Does drug resistance mutation have any relationship with HBV genotype and disease progression?How does the rt181 mutation affect the S gene?Does this site variation have any clinical impact?Based on these ideas we have established this research.This study is divided into the following three parts:PART ?:Analysis of pre-existing drug resistance in patients with chronic hepatitis B virus infectionPART ?:Sequence Analysis of HBV Polymerase Region Gene in Chronic Hepatitis B Virus Infected Patients with Virological BreakthroughPART ?:Effect of rt181 mutation on S gene in patients with chronic hepatitis BPART I:Analysis of pre-existing drug resistance in patients with chronic hepatitis B virus infectionBackgroundHow to prevent or delay the progression of patients with chronic hepatitis B is a serious problem for liver disease researchers.Because so far people still have not found a way or weapon to absolutely eliminate the hepatitis B virus.For the treatment of chronic hepatitis B,antiviral therapy is currently the only effective way to minimize viral replication and delay progression.Among antiviral drugs,the antiviral effect of NAs on CHB has been confirmed.However,all oral NAs,including lamivudine(LAM),adefovir(ADV),telbivudine(LDT),and entecavir(ETV),can present varying degrees of resistance during treatment.Therefore,the presence of drug-resistant strains poses great difficulties in the treatment of hepatitis B.But are these drug-resistant strains present at the beginning of the drug or are they induced by the drug?Patients with chronic hepatitis B also have a primary non-response during the course of taking anti-viral therapy with NAs.What is the reason for the primary non-response?Is there a pre-existing resistance mutation in the hepatitis B virus itself?If pre-existing drug resistance mutations exist,how does this pattern of pre-existing drug resistance have to do with the different stages of liver disease progression?Does drug resistance have any relationship with genotype?These issues have not yet been clarified.Whether or not genetic drug resistance testing should be performed before antiviral therapy is also mixed.In order to clarify the above problems,we have established this study to understand whether there is pre-existing drug resistance in chronic HBV-infected patients,the relationship between pre-existing drug resistance and genotype,and the relationship between pre-existing drug resistance and disease progression.Objective1.To understand the distribution of HBV genotypes in patients with chronic hepatitis B virus infection in Xuzhou,Jiangsu.2.To observe the prevalence of drug resistance and the pattern of drug resistance mutation in chronic hepatitis B virus infection.3.To observe the relationship between pre-existing drug resistance and HBV genotype and disease.MethodsCollection of 357 patients with chronic HBV infection without antiviral therapy,automatic biochemical analyzer for liver function,chemiluminescence microparticle immunoassay for quantitative determination of HBV-M,using PCR product direct sequencing to detect HBV DNA polymerase(P)region gene sequence,the sequencing results were compared on NCBI's Genotyping tool.All measurement data were expressed as mean±standard deviation(x×s).The variance homogeneity test was performed first,followed by one-way ANOVA and pairwise comparison.The incidence of variation between different genotypes was compared using the X2 test.The test level was bilateral a = 0.05,P<0.05 was considered statistically significant.ResultsPre-existing drug resistance variants associated with lamivudine were found in 357 patients with chronic hepatitis B virus infection,8 patients(3.7%)in the chronic hepatitis B(CHB)group and 13(11.7%)in the cirrhosis(LC)group.In the liver cancer(HCC)group,6 patients(21.4%),the pre-existing drug resistance mutations associated with adefovir dipivoxil were detected in 10 patients(4.6%)in the CHB group,15 patients(13.5%)in the LC group,and HCC group 4 Case(14.5%);pre-existing drug-resistant variants associated with lamivudine and adefovir dipivoxil were detected in 2 patients(0.9%)in the CHB group,1 patient(0.9%)in the LC group,and 1 patient in the HCC group(3.6).%).The prevalence of drug-resistant mutations associated with lamivudine and the prevalence of pre-existing drug resistance mutations associated with adefovir dipivoxil were statistically significant(P<0.01).Moreover,the above-mentioned drug resistance mutations occurred in the C genotype,and no occurrence of pre-existing drug resistance was found in the B genotype.The detected resistance patterns were mainly rtS213T(4.8%),rtV214A(3.6%),and rtN/H238T/D(3.1%).The primary drug resistance variants(rtA181T/V,rtM204V/I/S,rtN236T,and rtM250I/V)had lower incidence,while the compensation variants such as rtV173L,rtV214A,rtQ215S,and rt N/H238T/D were higher.Conclusions1.The HBV genotypes of chronic hepatitis B infected people in Xuzhou,Jiangsu Province are mainly C and B.2.There are pre-existing drug resistance of lamivudine and adefovir dipivoxil in chronic hepatitis B virus infected patients,pre-existing drug resistance mode is mainly Compensation variation.3.The occurrence of pre-existing drug resistance is related to the progression of liver disease,and the incidence of pre-existing drug resistance in endstage of liver disease is higher.4.Pre-existing drug resistance is related to genotype.HBV C genotype is more susceptible to drug-resistant variation than B-type.PART II:Analysis of drug resistance mutations in HBV polymerase region in chronic hepatitis B patients with virological breakthroughBackgroundIn the anti-viral treatment of chronic hepatitis B,the efficacy of nucleoside(acid)analogues(NAs)has been affirmed.However,all oral NAs,including lamivudine(LAM),adefovir dipivoxil(ADV),and telbivudine(LDT),may present different degrees of resistance during treatment.Sex.Although the initial selection of entecavir(ETV)resistance rate is relatively low,and patients who have developed lamivudine resistance have been treated with entecavir for 5 years,their resistance is as high as 51%,and lamivudine-resistant HBV merger HIV-infected people also experienced virological breakthroughs during the use of TDF.Therefore,the presence of drug-resistant strains poses great difficulties in the treatment of chronic hepatitis B.Recent prospective studies have shown that antiviral drug resistance increases the risk of hepatocellular carcinoma in patients with decompensated hepatitis B cirrhosis,especially in patients who have failed to rescue treatment.In order to avoid the occurrence of drug resistance,the guidelines recommend tenofovir and entecavir as the first choice for antiviral therapy in NAs.However,due to the late listing of TDF in China(until it was not listed in July 2014),and because of economic reasons,many people with chronic hepatitis B often use ADF or LAM or a combination of two drugs in the initial antiviral.Application,therefore,these people need to closely observe the antiviral treatment process,regularly review HBVDNA,once the virological breakthrough is found,detect the drug resistance mutation as soon as possible,accordingly adjust the antiviral treatment plan in time,control the progress of the disease,reduce the virus breakthrough and even rebound The re-strike caused by the body is very important.In addition,patients with virological breakthroughs have no clear conclusions about the genetic mutations associated with HBV genotypes and disease progression or the antiviral drugs used.For this we have set the following research.Objective1.To investigate the genetic resistance variation of chronic HBV infection in virological breakthroughs.2.To explore the relationship between genetic mutations in chronic HBV infections and HBV genotypes and conditions.MethodsA total of 141 serum samples from patients with viral rebound were collected,and the Polymerase gene of HBV was sequenced directly using PCR products.Automatic biochemical analyzer for detecting liver function,chemiluminescence microparticle immunoassay for quantitative determination of HBV-M.Among the recruited patients,59 cases were diagnosed with CHB,68 cases of liver cirrhosis and 14 cases of primary hepatocellular carcinoma.Adefovir dipivoxil(ADV)was administered for 51 cases,lamivudine(LAM)or telbivudine(LDT)for 39 cases,entecavir(ETV)for 10 cases and LAM or LDT switching to ADV for 41 cases.Comparison of mutation rates between groups were compared by x2 test.Results1.There was no significant difference in age,sex,ALT level and HBV DNA load between the different treatment groups(P>0.05).2.Distribution of HBV genotypes.The HBV genotypes detected in 141 patients with virological breakthrough chronic HBV infection were mainly C-type 138/141(97.9%),and only B cases(3%)(2.1%).No other genotypes were detected.3.Distribution of drug resistance variation patterns in different treatment groups.The drug resistance mutation patterns detected in the ADV treatment group were mainly rtA181T/V or rtS213T single-point variation and multi-point mutation pattern containing rtA181T/V or rtS213T.The mutation pattern detected by the LAM/LDT treatment group was mainly rtL180M rtM204I/V./S and rtM204I/V/S or multi-point mutation pattern containing 204 sites.the drug resistance pattern in the ETV treatment group was simultaneous presence of multi-site mutations containing rtL180M rtM2041/V/S,The type of drug resistance variant is a complex variation pattern in which multiple sites are simultaneously resistant to ADV and LAM.The most frequent frequency of resistance mutations was rtM204I/V/S(35.5%),followed by rtL180M(22.7%),followed by rtA181T/V(19.1%).4.The relationship between drug resistance mutations and therapeutic drugs.Comparing the frequency of drug resistance mutations in different antiviral treatment groups,30 cases(30/41,82.1%)showed resistance mutations in the LAM/LDT sequential ADV group,and 28 cases showed resistance mutations in the LAM/LDT group(28/39,71.8%),there were 20 cases of drug resistance mutations in the ADV antiviral treatment group(20/51,42%),and 3 cases(3/10,30%)in the ETV group.There was a statistically significant difference in the occurrence of drug resistance between the groups,P<0.01.5.The relationship between drug resistance mutation and disease.Among the 141 patients with chronic HBV infection,59 patients in the CHB group,30 patients with drug resistance(30/59,50.8%),68 patients in the LC group,40 patients with drug resistance(40/68,58.8%),PLC There were 14 cases in group,and 12 cases showed resistance mutations(12/14,85.7%).It seems that the proportion of drug resistance increases with the progress of the disease,but there is no statistical difference,P = 0.059.Conclusions1.The incidence of drug-resistant mutations was highest after lamivudine/tebivudine sequential adefovir dipivoxil treatment,followed by lamivudine or telbivudine,and entecavir resistance was the lowest.2.The incidence of drug resistance mutations in virological breakthroughs is related to the initial selection of nucleoside drugs and has nothing to do with disease progression.3.The incidence of drug-resistant mutations in virological breakthroughs is related to HBV genotypes,and type C is more susceptible to drug resistance than type B.PART ?:Effect of rt181 mutation on S gene sequence in patients with chronic hepatitis BBackgroundThe efficacy of nucleoside(t)ide analogues(NAs)drugs against hepatitis B virus has been recognized by the medical community.However,with the prolongation of NAS treatment time,there will be different degrees of viral resistance,which will affect its antiviral treatment effect.The 181th amino acid variation in the reverse transcriptase region presents a new nucleoside antiviral therapy."Challenge" because this site mutation causes HBV to be resistant not only to adefovir dipivoxil,but also to lamivudine,entecavir,telbivudine,and tenofovir.The degree of decline,and this site variation can be induced not only during the treatment of adefovir dipivoxil,but also during the treatment of telbivudine.The presence of drug-resistant variants in patients who have never been treated with antiviral therapy is known as pre-existing drug resistance,and our previous study found that the rtA181V/T variant is also present in patients with chronic hepatitis B who have never been treated with antiviral therapy.At present,the results of in vitro experiments suggest that the rtA181T/V variant virus has a certain degree of resistance to most nucleoside(acid)drugs.Since the S gene of HBV(nt155-835)completely overlaps with its reverse transcriptase RT gene(nt131-1161),when a certain mutation occurs in some base sites in the RT region,the corresponding site in the S region is changed.As a result,the codons formed by the transcription of the S gene are changed,which in turn leads to a corresponding change in the HBsAg protein encoded by the codon.The rtA181T mutation pattern can also cause truncation or substitution mutations in HBsAg,such as the rtA181T/sWn172*variant and the rtA181T/sW172L variant.This defective HBsAg with a truncation/replacement mutation can cause cancerous changes in hepatocytes and can also result in a decrease in serum HBsAg levels.In the clinic,we observed that rt181 mutation occurs not only in patients with liver cancer,but also in patients with chronic hepatitis B.So is there any difference between pre-existing drug resistance in patients with chronic hepatitis B and adefovir dipivoxil-induced drug resistance?Is there any relationship between rtl81 gene mutation and S region gene mutation?What is the impact on the expression of HBsAg?Does this mutation have any relationship with genotype?Based on this idea,we set up this research.Objective1.To understand the relationship between pre-existing drug resistance variation and genotype in patients with chronic hepatitis B(CHB).2.To understand the similarities and differences between pre-existing drug resistance and adefovir dipivoxil-induced resistance mutations.3.To explore the effect of rt181 mutation on the S region gene sequence in patients with chronic hepatitis B.4.To learn about the effect of Rt181 mutation on serum HBsAg levelMethods298 patients with CHB were enrolled,including 226 CHB patients with no anti-viral treatment and 72 CHB patients who had a poor antiviral treatment with adefovir dipivoxil or virological breakthrough.Peripheral blood was collected and using automatic biochemical analyzer to detect the liver function,and the HBV-M was quantitatively determined by chemiluminescence microparticle immunoassay.The PCR product was directly sequenced in HBVP gene sequence,and the specific primers were designed to amplify the S region and perform gene sequence analysis.The count data were analyzed by x2 test;the quantitative data were expressed as mean±standard deviation(x± s),and the statistical method was analyzed by one-way ANOVA.The test level was bilateral a=0.05,P<0.05 was considered statistically significant.Results1.298 patients with CHB,including 281(94.3%)in type C and 17(5.7%)in type B.2.226 CHB patients who were not treated with antiviral drugs,211 were C genotypes and 15 were B genotypes.16 cases(7.1%)of pre-existing drug-resistant mutations were detected,and the genotypes were all C-type.Among them,there were 1 case of rtV 207L and 5 cases of rtS213T related to lamivudine resistance;8 cases related to adefovir resistance,1 case of rtA181T,3 cases of rtV214A,rtN/H 238D/S/T 4 cases.1 case of rtV207I rtN/H 23 8D associated with lamivudine and adefovir resistance,1 case of rtS213T rtN/H 238S.No resistance mutations were detected in the B genotype.3.72 patients with CHB treated with adefovir dipivoxil,70 were genotypes and 2 were B genotypes.There were 30 cases(41.7%)with drug resistance mutations,including 1 case related to lamivudine resistance,rtS213T,and 25 cases related to adefovir resistance,7 cases of rtA181T.There were 10 cases of rtA181V,2 cases of rtA181T rtN236T,1 case of rtA181T rt238T,2 cases of rtA181V rt238S,1 case of rtV 214A and 2 cases of rtN 23 6T.The mutations related to lamivudine and adefovir resistance were detected.For example,rtA181T rtV207I 1 case,rtA181T rtV2071 rtN236T 1 case,rtS213T rtV 214A 1 case,rtN 236T rtM 250L 1 case.One of the above cases of genetic variation was B genotype,which was rtN 236T mutation,and the rest were C genotypes.4.There was no significant difference in age,sex and HBV DNA load between the two genotypes of CHB patients(P>0.05).5.There were 5 patients with rtA 181T mutation and 2 patients with rtA 181T rtN236T.The corresponding amino acid at position 172 of the S region was changed from tryptophan(Trp,W)to leucine(Leu,L),ie rtA181T/sW172L.One patient with rtA181T mutation had a mutation in the base of sw182 amino acid to the stop codon TGA.Three patients with rtA181T mutation and three other patients with rtA181T multi-site variation(rtA181T rtV207I,rtA181T rtV207I rtN236T and rtA181T rt238T)mutated the base of the 172th amino acid sequence to a stop codon(TGA),resulting in S The truncation mutation of the region,ie rtA181T/sW172*;10 patients with rtA181V mutation and 2 patients with rtA181V rt238S,the 173th amino acid of S region changed from leucine(Leu,L)to phenylalanine(Phe,F),ie rtA181V/sL173F.6.In 298 CHB patients,2 Pre-S2 start codon mutations(ATG?AT A)were detected.Both patients were rtA181 V variants.7.There was no significant difference in the age and serum viral load between the RtA181T variant group and the rtA181V variant group(P>0.05).Serum HBsAg levels were significantly lower in the rtA181T and rtA181V variants than in the unvariant group(P<0.01).Conclusions1.Pre-existing drug resistance in CHB patients.2.HBV C genotype is more susceptible to drug resistance than B genotype.3.Pre-existing drug resistance mutantion is different from ADV-induced drug resistance mutantion,the main patterns of drug resistance mutations induced by ADV are primary resistance mutations,while pre-existing drug resistance mutations are mainly secondary resistance mutations.And the prevalence of Pre-existing drug resistance mutantion is lower than that of adefovir dipivoxil induced.4.The risk of rtA181T/V is higher when there is a virological breakthrough or poor treatment in the treatment of adefovir dipivoxil.5.HBV rtA181T variants are accompanied by variations in sW172L or sW172*.The rtA181V variant is accompanied by a variation in sL173F and may be accompanied by a termination mutation in the Pre-S2 initiation codon.6.The HBsAg level of CHB patients with rtA181 mutation decreased.