Analyses Of Clinical And Phenotpical Features Of RtA181T Mutation-causative Truncated S Protein-type And Non-truncated S Protein-type Mutations Of Hepatitis B Virus

Objective:When patients with hepatitis B virus chronic infection?CHB?were treated with nucleos?t?ide analogs?NAs?in clinical practice,HBV may emerge drug-resistance-associated mutations in reverse-transcriptase?RT?region such as rtA181 T which may consequently cause mutation in overlapping S region.The currently aimed to clarify clinical occurrence and drug-resistant features of the two-type rtA181 T mutations,i.e.,rtA181T/sW172stop?*?that causes truncated S-protein and rtA181T/sW172non-stop mutation that causes non-truncated S-protein.Methods:Total of 22,009 serum samples from individual NAs-experienced HBV-infected patients were collected from 2007 to 2016 in Research Center for Clinical and Translational Medicine,PLA 302 Hospital,Beijing.The characteristics of the HBV mutants with patients' clinical features and antiviral schedules were analyzed.Serum samples were collected,and HBV RT and S regions were amplified by nested PCR followed direct sequencing.Clinical features of patients with rtA181 T mutation in the large cohort of patients including the A181 T causative sW172*and sW172non-stop mutations were analyzed.In addition,serial serum samples were collected for the study from four long-term observed patients who were detected with both rtA181T/s W172*and rtA181T/sW172non-stop mutations during observation.Patient serum samples were collected and HBV DNA was extracted,HBV RT and S regions were amplified by nested PCR and Drug-resistant mutations were analyzed by cloning sequencing;Serum sample were collected from rtA181T/sW172non-stop mutation patients and the subtype of HLA-A2 was identified by nested PCR assay.The HBV RT genes harbouring wild-type or ten relevant rtA181T/sW172 mutants isolated from the same patient's clone strain,were cloned into pTriEx-1.1-HBV vector to generate replication-competent amplicons for phenotypic analysis.The mutant and wild-type HBV genomic amplicons were transiently transfected into HepG2 cells and cultured in the presense or absence of NAs,the level of HBsAg and HBV DNA was measured.Drug susceptibility was determined by comparing half maximal effective concentration(EC₅₀)of a mutant to wild-type strain.Results:5.37%?1,182/22,009?of the patients' samples was detected rtA181 T mutation.rtA181T-causative sW172non-stop?sW172L/S?,sW172*/non-stop and sW172*mutations occupied 7.70%,9.39%and 82.91%,respectively.The patients with rtA181T/sW172non-stop mutants had a significantly higher HBV DNA level compared to those with rtA181T/sW172*mutants.The detection rate of rtA181T/sW172non-stop mutation had an increasing trend over time,i.e.,4.43%?27/610?during year 2007-2010,10.09%?34/337?during year 2011-2013,and 12.77%?30/235?during year 2014-2016 of total rtA181T-positive samples?P<0.05?.In the combined drug-resistant mutation pattern,44.33%?524/1,182?rtA181T-positive samples were concomitant with recognized drug relevant resistance mutations,including 325 with adefovir relevant resistance mutations rtA181V/N236 T,57 with lamivudine relevant resistance mutations rtM204V/I,99 with entecavir relevant resistance mutations rtM204V/I plus rt184/202/250 substitutions,and 43 with multidrug-resistantmutationsrtA181V/N236T+M204V/I±184/202/250 substitutions.Adefoivr-resistant mutations more frequently coexisted with rtA181T/sW172non-stop mutation than rtA181T/sW172*mutation?42.86%vs.24.59%,P<0.001?.The HLA-A2 subtype was detected in 43.07%?87/202?of the rtA181T/sW172non-stop mutation patients' samples.Phenotypic analysis,rtA181T/sW172S+rtN236T and rtA181T/sW172L+rtN236T strains exhibited higher replication capacity and less adefovir susceptibility than that of rtA181T/sW172*+rtN236T strain?98.02%and 85.5%vs.42.1%in replication capacity,and 7.38-fold and 5.49-fold vs.3.69-fold in half maximal effective concentration of wild-type strain?;rtA181T/sW172L+rtS202G+rtM204V strain exhibited higher replication capacity and less entecavir susceptibility than that of rtA181T/sW172*+rt S202G+rtM204V strain?50.98%vs.34.49%,524.00-fold vs.69.33-fold of wild-type strain?.Conclusions:In clinical,HBV rtA181T/sW172non-stop mutation had an increased occupation in total rtA181 T mutations in recent years.rtA181T/sW172non-stop mutation may confer higher viral replication capacity and less drug susceptibility to adefovir-and entecavir-resistant HBV mutants and influenced clinical presentation of the NA-resistant patients than rtA181T/sW172*mutation.