| BackgroundHepatitis B virus(HBV)infection remains a major public health problem worldwide,and it is one of the serious infectious diseases that need to be controlled in China.Shandong Province,which is located in the east of China with huge population and strong economy,has made tremendous progress and become an intermediate HBsAg prevalence area by continuing to make universal infant HBV immunization as the main prevention and control strategy.However,the number of HBV infected individuals remains large for the large population base in Shandong Province.Thus,the situation of HBV prevention and control is still grim in Shandong Province.With the development of molecular epidemiology and molecular evolution,in recent years,it provides new methodological support for the research on etiology characteristics,gene mutation and evolutionary origins of HBV.Shandong Province still faces several challenges in the process of entering HBV low prevalence area.For instance,previous studies have mainly focused on comparisons of HBV infection rates before and after the use of hepatitis B vaccine,and scarcely on the sustained change in HBV prevalence after widespread hepatitis B vaccination;whether long term universal infant HBV immunization could increase the prevalence of hepatitis B vaccine escape variants;whether widespread use of antiviral drugs could increase the prevalence of drug-resistance mutation;the origins,spatial and temporal dynamics of HBV is not clear.Researches on above unsolved issues are important to explore the epidemic features,molecular evolution,and the influence of population size and migration on pathogenicity of HBV in Shandong Province.ObjectivesTo identify the latest epidemiology and changing trend of HBV infection by comparing the results of 1992,2006 and 2014,basing on serosurvey among population aged 1-59 years in communities in Shandong Province.To explore the genetic characteristics,mutation and recombination of HBV,and the changing trend of molecular epidemiology by whole-genome and S gene sequencing of HBsAg positive samples obtained in 2014.To analyze the origins,spatial and temporal dynamics of HBV using Bayesian Markov Chain Monte Carlo(MCMC),basing on representative whole-genomes of global HBV genotype C.Methods1.We conducted a serosurvey and collected blood samples by a multi-stage,stratified,random sampling method among the population aged 1-59 years in 12 counties in Shandong Province,China in 2014.The blood samples of participants were collected,Hepatitis B surface antigen(HBsAg),antibody against HBsAg(anti-HBs),and antibody against hepatitis B core antigen(anti-HBc)were tested by Enzyme-Linked Immunosorbent Assay(ELISA).Hepatitis B vaccination and the prevalence of HBV biomarkers were analyzed by constructing appropriate sampling weights,and were compared with the results in 1992 and in 2006 in Shandong Province.2.HBV DNA was amplified from HBsAg positive samples by PCR and complete genome and S gene was sequenced.HBV genotypes and subgenotypes were determined depending on S gene and complete genome phylogenic analysis respectively.All the sequences in this study were screened for relevant mutations by comparing with reference HBV genome sequences of different genotypes.Recombination analysis was performed within the acquired HBV sequences.HBV genotypes,mutations and recombination events from HBsAg positive samples in 2014 were compared with those in 2006.3.Full-length genomes of HBV genotype C isolated worldwide from Genbank were selected,which were integrated with full-length genomes from this study as molecular evolution analysis datasets.Origin and evolution was analyzed by Bayesian Markov Chain Monte Carlo(MCMC).Spatial and temporal dynamics of HBV were analyzed by Bayesian Stochastic Search Variable Selection(BSSVS).Results1.This investigation involved 12 counties,which accounted for 8.57% of all inv counties in Shandong Province.Overall,5,580 individuals participated in the investigation.Of those,5,531 blood samples were collected and tested.Finally,5,528(99.07%)were included in analysis.Eighty seven individuals with HBsAg positive were screened among 5,528 individuals in 2014.Overall,the prevalence of HBsAg was 2.49%(95%CI: 1.79%-3.19%),which was lower than that in 1992(6.4%)and in 2006(3.39%).The prevalences of HBsAg increased among persons aged 20-24 years and 40-44 years in 2014 compared with 2006(t=0.44,0.18,all P>0.05).The prevalence of anti-HBs was 48.27%(95%CI: 45.48%-51.07%)in 2014,which decreased with age(F=235.95,P<0.01).The prevalence of anti-HBc was 22.56%(95%CI: 20.14%-24.97%)in 2014.The prevalence of anti-HBc showed moderate increases in students and farmers in 2014 compared with 2006.The prevalence of HBsAg,anti-HBs and anti-HBc was 2.10%(95% CI: 0.97%-3.23%),47.72%(95%CI: 41.68%-53.75%)and 24.40%(95%CI: 18.50%-30.30%)respectively among women at child-bearing age in 2014.The prevalence of HBV susceptibility was 47.55%(95%CI: 41.63%~53.47%).The prevalence of HBsAg among vaccinated persons was 1.26%(95%CI: 0.67%-1.85%),which was significantly lower than that among unvaccinated persons(3.98%).2.Among 5,528 subjects included in the final analysis,3,628 and 3,289 subjects received at least one dose and three doses of HepB respectively.Coverages with one dose and three doses of HepB were 46.34%(95%CI: 40.76%-51.92%)and 38.21%(95%CI: 35.84%-40.58%)respectively.The coverages with one dose and three doses of HepB were above 98% and above 97% respectively among children under 15 years old.Both the coverages with one dose and three doses of HepB decreased gradually with age among individuals aged 15-59 years.The HepB coverage for each age group in 1992 was significantly lower than that in 2006 and in 2014.The HepB coverage among individuals aged 10-59 years in 2014 was slightly higher than that in 2006.3.Nine HBV complete genome sequences and 70 HBV S gene sequences were acquired from 87 samples with HBsAg positive.Of the 70 S gene sequences,genotype C and D accounted for 95.71%(67/70)and 4.29%(3/70)respectively,and subtype adrq+ accounted for 94.29%(66/70).T47A/E/K/M in the S region of HBV DNA was the predominant mutation,and it was identified in 15.71%(11/70)of subjects.The overall prevalence of mutation in the “a” determinant region was 12.86%(9/70),which was slightly lower than that in 2006(14.71%).The predominant mutation amino acid(AA)changed from I126 S in 2006 to T131 P in 2014.Pre-C G1896 A mutation was present in 1 sample.A1762 T and G1764 A BCP double mutations were present in another sample.L116 V,P36T/A,L/P37/I/S mutations in X region of HBV were present in 4 samples,3 samples and 3 samples respectively.V5 M,F34L,S43 A and S101 A were observed in 2 samples.None of reported drug-resistant variations was observed.In addition,none of recombinant events between different genotypes was observed.4.In total,2,541 complete genome sequences of HBV genotype C with isolated time and geographic location were downloaded from Genbank(http://www.ncbi.nlm.nih.gov/ genbank/),98 of which were included in the final analysis.The predominant genotype was subgenotype C2 in the 98 complete genome sequences(48.98%).The most recent common ancestor(tMRCA)of global HBV genotype C was dated to Anno Domini(A.D.)567 in Australia,with 95% highest posterior density interval(HPD)being from Before Christ(B.C.)1474 to A.D.1683.HBV subgenotype C4 diverged from the tree root earlier than subgenotype C1-C3 and circulated in Australia separately,with tMRCA dated to A.D.1008(95% HPD: B.C.406-A.D.1786).The estimated time for the origin of HBV genotype C from Shandong Province,China was dated to A.D.914(95%HPD: B.C.625-A.D.1754),entering Shandong from Indonesia in 1279.Population dynamic analysis showed that an increase in the effective number of infections grew from the 1660 s,followed by a significant increase from the 1810 s until the 1860 s,and then kept at a steady state,and decreased from the 1990 s until 2016.Conclusions1.The prevalence of HBsAg continued to decrease and the prevalence of anti-HBs had an increase since 2006,which suggested that universal infant HBV immunization as well as other prevention and control measures mantain effective.Young students and farmers became the high risk populaiton of HBV infection.The prevalence of HBsAg for women at child-bearing age approached the low prevalence level.However,the prevalence of HBV susceptibility was high.The HepB coverage continued to increase among persons aged 1-59 years in Shandong Province,China from 2006 to 2014.The HepB coverage mantained high among children aged 1-15 years since 2006.However,the HepB coverage was low among adults.The high risk population and the route of transmission has changed,which suggested that surveillance on emerging HBV infections should be strenthened,and HepB should be provided for young students,farmers and women at child-bearing age.2.HBV genotype C was the predominant genotype,with emerging of minor genotype D.Although the predominant mutation changed from I126 S in 2006 to T131 P in 2014,the prevalence of mutation was stable in the “a” determinant region.None of reported drug-resistant variations and recombinant events between different genotype was observed.It suggested that universal infant HBV immunization and antiviral therapy do not cause significant changes on molecular epidemiology of HBV.4.The global human HBV genotype C originated 1400 years ago.The originated time of subgenotype C4 was earliest among C1-C4.HBV genotype C entered Shandong province from Indonesia in 1279.There was a significant increase on the effective number of infections in the first half of 19 th century and decreased after 1990 S,which possibly linked with implementation of a newborn vaccination program worldwide. |
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