| BackgroundChronic hepatitis B infection,caused by hepatitis B virus(Hepatitis B virus,HBV),is a major public health threat in China.Hepatitis B vaccine is the most effective means to prevent and control HBV infection and transmission.Since hepatitis B vaccine was included in children's planned immunization in 2002,the carrying rate of chronic HBV surface antigen(Hepatitis B virus surface antigen,HBsAg)in Chinese population has decreased significantly.However,more and more scholars have reported that the emergence of vaccine escaped HBV(ve-HBV)has become an important obstacle to further improve the effectiveness of hepatitis B prevention and control recently.Studies have shown that in the main hydrophilic region(MHR)of HBsAg,the variation of some important amino acids(aa)such as G145R is a common and important cause of ve-HBV.In addition,the mutations in E2G,L21S and L98V outside MHR also affect the replication and secretion of the HBV.In order to provide more scientific basis for the prevention and control of HBV infection,we firstly analyze the molecular evolution trends of ve-HBV associated with mutations in/outside of MHR in China from 2000 to 2016 in the present study.Subjects and methods(1)According to the results from previous studies,the HBV with I/T126A/N/I/S,Q129H/R,M133L,K141E,P142S,D144A/E and G145R/A mutations in the MHR region were defined as ve-HBV.In the present study,528 ve-HBV collected from GenBank were isolated from China between 2000 and 2016.MEGA 6.06 was used to analyze paired distance(p-distance),JModeltest was used to analyze nucleotide substitution model and site rate change model.The phylogenetic tree and molecular evolution rate were analyzed by Bayesian Markov chain Monte Carlo(MCMC)method of BEAST v1.6.1.Tracer v1.6.1 was used to show the molecular evolution rate of ve-HBV and Bayesian skyline plot(BSP),and HYPHY program was used to analyze the selection pressure of amino acid sites.(2)In this study,231 and 348 HBsAg positive plasma samples were collected from non-chronic HBV infected population and chronic hepatitis B patients in Suzhou,respectively.HBV surface antibody(HBsAb)positive serum was derived from hepatitis vaccinated children(HBsAg,HBcAb,HBeAg and HBeAb are negative).Firstly,the virus sequence in HBsAg positive plasma was isolated by nested PCR and sequenced.Secondly,the neutralization ability of HBsAb positive serum to different mutant HBV strains outside MHR region was analyzed by ELISA-based neutralization test in vitro.Finally,Lasergene was used to analyze the changes of B cell and T cell epitopes of HBV mutants.Gamier-Robson,Chou-Fasman and Phyre 2 were used to predict the secondary and tertiary structural characteristics of HBV mutants.(3)SAS 9.4 was used to collate and analyze the data.The mean±standard deviation was used for the continuity variables of normal distribution,the rank sum test was used to compare the neutralization rate between the two groups,and the classification variable was expressed as rate(%),the correlation between the cell epitope mutation level and the neutralization experiment results were analyzed by Logistic regression method.All P values were based on a 2-sided test and a significance level of 0.05.Results(1)Among the 528 ve-HBV,there were 215 and 313 strains of genotype B and genotype C ve-HBV,respectively.The results showed that the genotype B ve-HBV differentiated in 1997(95%CI:1987-2005),genotype C ve-HBV differentiated in 1976(95%CI:1955-2003).The genotype B ve-HBV evolved faster than the genotype C ve-HBV(2.103×10-3/year vs 1.083×10-3/year).In addition,the p-distance of the genotype C ve-HBV was 0.0291±0.0169,which was significantly higher than that of the genotype B ve-HBV(0.0145±0.0121,t=131.02,P<0.05).(2)BSP analysis showed that the effective population size(ESP)of genotype B ve-HBV slightly increased until 2007 after the overall trend remained stable from 1996 to 2003,then the population declined in 2011,remained constant,and expanded in 2016 again.For genotype C ve-HBV,the results showed that EPS gradually increased from 1996 to 2003,and relatively stable from 2003 to 2011,after which the population obviously decreased and expanded between 2011 and 2013,finally a constant size was maintained.The EPS of all ve-HBV is similar to genotype C ve-HBV.On the whole,the population fluctuation of genotype C ve-HBV are larger than that of genotype B ve-HBV,both EPS of genotype B and genotype C ve-HBV are rising and then maintaining a relatively stable evolutionary trend.(3)The results showed that among the genotype B ve-HBV,four sites were positively selected(A5T/S,L21S,T/A126S and T/N131I/A).While among genotype C ve-HBV,there were 13 positive selection sites(N3S,T5A,G10Q/R/E,L21S,T47K/A/V,L98V/P,I/S126N/V/T,Q129H/R/L,T131P/I/N/A,G145A/R,L175S/F,L213I/S,V224A/G),suggesting that mutations at these sites have a tendency to form a potential epidemic.(4)A total of 183 full-length sequences of HBV/S region were amplified in 579 HBV-positive plasmas,of which 95(51.91%)were mutant HBV strains outside MHR.Among the mutant HBV strains outside MHR,20 and 75 were genotype B and genotype C HBV,respectively.In addition,there were 276 amino acid mutations(1.29%)involving 70 sites,and the site mutation rate was 30.97%(70/226).The site with the highest mutation frequency was locatedPerinatal at T47A/K(26.32%),followed by M/L213I and Y200F/L/C,and the corresponding mutation rates were 22.11%and 20.00%,respectively.(5)All of genotype B HBsAg-positive plasma with mutant HBsAg outside MHR could be neutralized by HBsAb-positive serum induced by hepatitis B vaccine,while 34 genotype C HBsAg-positive plasma could not be neutralized by HBsAb-positive serum induced by hepatitis B vaccine(45.33%).Furthermore,our results showed that the mutation rate of the B-cell epitope,aal-10 region in the non-neutralization group was 2.73 times(95%CI:1.06-7.01)more than those in the neutralization group.Moreover,N3S or T5A mutations in aal-10 region not only altered the epitope structure of B-cells epitope of antigen,but also affected the three-dimensional structure of aal-10.(6)In 2000-2016,the EPS of the HBV strains containing N3S or T5A mutations in China,increased rapidly in 2005 and then remained stable.However,in 2011,there was a rapid decline and then rebounded.After 2015,these strains showed a downward trend.In general,HBV with N3S or T5A mutations showed an upward trend in evolution from 2000 to 2016.Conclusions(1)Among ve-HBV in China during 2000-2016,the genetic diversity of genotype C ve-HBV was much higher than those in genotype B ve-HBV.However,genotype B ve-HBV evolved more rapidly than those in genotype C ve-HBV.Additionally,the EPS dynamics were genotype-specific.The population of genotype C ve-HBV fluctuated more than those in genotype B ve-HBV.(2)Among HBsAg of genotype C HBV,N3S or T5A mutations in aal-10 region can affect the neutralizing potency of vaccine-induced HBsAb because these two mutation changes secondary and tertiary structure of aa1-10 region.In addition,between 2000 and 2016,the mutations of N3S and T5A had formed positive selection,and the effective population size of HBV containing these two sites showed an increasing evolutionary trend,suggesting a tendency to form a potential epidemic. |
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