Study On The Primary Drug Resistance Mutation Of Hepatitis C Virus In Chinese Voluntary Blood Donors And The Mechanism Of S Protein Transmembrane Domain Mutation Affecting The Occurrence And Development Of Latent Hepatitis B Virus Infectio

Hepatitis C virus(HCV)infection is a challenge for global public health.Although China displays low prevalence rate of HCV,it still endures a heavy burden of this disease because of its large population base.HCV genome is 9.6 kb,including a large open reading frame and two untranslated regions.Ribosomes translate the HCV genome into a polyprotein of approximately 3000 amino acids through the internal ribosomal entry site of the 5’ untranslated region(5’ UTR).The polyprotein is separated into different viral proteins under the cleavage of host and viral protein enzymes.Among the viral proteins,non-structural proteins NS3,NS5A and NS5B are the targets for direct-acting antiviral agents(DAAs).The treatment regimen of DAAs is bioeffective and highly tolerated by patients,raising hope for the cure of HCV.However,resistance-associated substitutions(RASs)may influence the DAAs and block the sustained virologic responses(SVR),leading to the failure of DAA treatment.Understanding the prevalence of HCV and the occurrence of RASs in specific populations in China can provide targeted and effective treatment for HCV infection.In this part,we designed pan-genotype primers for various subtypes of HCV,targeting conserved regions(5’UTR and NS5B)of HCV genome.After amplifying the target fragment,sequencing,and mapping with the evolutionary tree,we acquired the primary subtyping results of 262 HCV RNA positive samples from Chinese voluntary blood donors.Based on the primary subtypes,we designed subtype-specific primers to amplify NS3 and NS5A regions.With the sequencing results of DAAs targets,we were able to calculate the occurrence rates of RASs,codon usage bias and corresponding genetic barriers.The results revealed that 1b and 2a were the two main subtypes in China,followed by subtype 6a,3b,3a,and 1a.Among them,subtype lb was the most popular subtype in all regions,and the proportion of subtype 2a was relatively high in blood donors from northern China.In the analysis of RASs,we found subtype 2a and 6a had limited RASs,and no evidence showed they influence the treatment of DAAs.Subtype 1b showed a higher RASs occurrence rate:in the NS3 region,substitutions(T54S,V55A,Y56F,Q80K/L,G122G/T,R117H/C,V170I and S174A)presented in 89.7%of the samples;in the NS5A region,substitutions(M28L,R30Q,P58L/S and Y93H)were observed in 22.1%of the samples.The most frequent RASs in NS3 and NS5A were S122G and Y93H,respectively.Moreover,the Y93H caused medium-to-high level resistance to all six commercialized NS5A inhibitors.Codon usage of these two substitutions showed that they both required only one transition to confer resistance.Their wildtype codons had lowest genetic barrier,which facilitated the generation of resistance mutants.This study showed the prevalence rates of different HCV subtypes in Chinese voluntary blood donors,investigated the frequency and genetic barrier of baseline RASs in NS3 and NS5A regions from these untreated blood donors.This study found lower genetic barriers facilitated the generation of resistance mutants and threated the efficacy of DAA regimens.The baseline RASs posed a great challenge to real-world DAA application.The results offered valuable information for DAAs optimization,and provided an experimental basis for the establishment of HCV treatment strategies.Occult HBV infection(OBI)is a special infectious status during Hepatitis B virus(HBV)infection.The definition of OBI is hepatitis B surface antigen(HBsAg)cannot be detected in blood by current detection assays,but HBV DNA can be detected in blood and/or liver tissues.Although it is believed that the interactions between viral and host factors lead to the occurrence of OBI,the underlying mechanism has not been elucidated.At present,there are many defects in OBI researches,such as small sample size,sporadic cases researches,limited research regions,lack of mechanism studies,etc.More comprehensive and in-depth exploration is needed to fill the gap and reveal the specific mechanism of OBI occurrence and development.HBsAg is an important component on outer envelope of HBV.Among HBsAg,S protein is encoded by 155835 nucleotides and includes 226 amino acids.S protein consists of four transmembrane domains(TMDs)and one major hydrophilic region(MHR).TMDs are mainly composed of hydrophobic amino acids.Mutations may change the properties of TMDs,make S proteins unable to anchor stably on the outer envelope of the virus,and affect the assembly and secretion of HBsAg as well as virus particles.There are few reports about the relationship between TMD mutations and OBI.In this part,we conducted sequencing analysis on 104 OBI samples and 524 HBsAg positive samples from 29 blood centers.We searched for high-frequency mutations in the TMDs of S protein in OBI population,constructed expression plasmids of TMD high-frequency mutations,and verified the possible molecular mechanism of OBI with in vivo and in vitro functional experiments.We found 22 high-frequency TMD mutations in genotype B OBI strains.Among them,5 mutations(G10R,C85R,F220C,F220Y and S174N+P178Q)can lead to impairment of HBsAg secretion;7 mutations(A5T,G7R,E164G,V168A,S174N,L175S and V168A+V177A)had accumulated intracellular HBsAg while extracellular HBsAg didn’t decrease compared to wildtype.This study chose C85R from TMD2,F220C and F220Y from TMD4 for further exploration.These three mutations exhibited lower extracellular HBsAg,while their intracellular HBsAg expressed at a high level.Protein structure predication showed the mutant HBsAg displayed changed hydrophilic properties and tended to accumulate in the phospholipid bilayer of cell membrane.Mutant HBsAg’s secretion disorder may induce OBI.On the other hand,V168A+V177A mutation from TMD3 expressed a large amount of HBsAg in both intracellular and extracellular level.It is a combined high-frequency mutation from OBI,but shows a different serological pattern from OBI.We also found the expression of extracellular and intracellular HBsAg in V168A+V177A was significantly higher than that in V177A(with one less mutation compared to V168A+V177A),and V168A+S174N+V177A(with one more mutation compared to V168A+V177A).Protein structure prediction showed the natural conformation of V168A+V177A was the most unstable,and it was inclined to transition into V177A or V168A+S174N+V177 A.Under the condition of OBI,these three mutations were prone to mix and coexistence.When V168A+V177A occurs with V177A and/or V168A+S174N+V177A,the extracellular HBsAg will be lower and the intracellular HBsAg will be higher,approaching the secretion disorder pattern of OBI.In summary,this study systematically studied the relationship between TMD mutations and OBI based on the sequencing results of OBI samples in China.This study clarified the effects of high-frequency TMD mutations,provided possible molecular mechanisms of their roles in the occurrence of OBI.Our findings will establish a theoretical and practical basis for the development of OBI and for the functional cure of chronic HBV infection.